Rac1
Bacterially expressed glutathione S-transferase fusion proteins containing Rac1 were used to identify binding proteins of this Rho family GTPase present in a bovine brain extract. Five proteins of 85, 110, 125, 140 and 170 kDa were detected, all of which were associated exclusively with guanosine 5'-[gamma-thio]triphosphate-bound Rac1, not with GDP-bound Rac1. The 85 and 110 kDa proteins were identified as the regulatory and catalytic subunits respectively of phosphatidylinositol 3-kinase. Several lines of evidence suggested that the 125 kDa protein is identical with Nck-associated protein 1 (Nap1). The mobilities of the 125 kDa protein and Nap1 on SDS/PAGE were indistinguishable, and the 125 kDa protein was depleted from brain extract by preincubation with the Src homology 3 domain of Nck to which Nap1 binds. Furthermore, antibodies to Nap1 reacted with the 125 kDa protein. Nap1 was co-immunoprecipitated with a constitutively active form of Rac expressed in Chinese hamster ovary cells. The observation that complex formation between activated Rac and PAK, but not that between Rac and Nap1, could be reproduced in vitro with recombinant proteins indicates that the interaction of Nap1 with Rac is indirect. The 140 kDa Rac-binding protein is a potential candidate for a link that connects Nap1 to Rac. The multimolecular complex comprising Rac, Nap1 and probably the 140 kDa protein might mediate some of the biological effects transmitted by the multipotent GTPase (Kitamura, 1997).
Rac1 small GTPase plays pivotal roles in various cell functions such as cell morphology, cell polarity, and cell proliferation. IQGAP1 from bovine brain cytosol has been identified as a target for Rac1 by an affinity purification method. By using the same method, a specifically Rac1-associated protein with a molecular mass of about 140 kDa (p140) was purified from bovine brain cytosol. This protein interacted with guanosine 5'-(3-O-thio)triphosphate (GTPgammaS).glutathione S-transferase (GST)-Rac1 but not with the GDP.GST-Rac1, GTPgammaS.GST-Cdc42, or GTPgammaS.GST-RhoA. The amino acid sequences of this protein revealed that p140 is identified as a product of KIAA0068 gene. This protein has been denoted as Sra-1 (Specifically Rac1-associated protein). Recombinant Sra-1 interacts with GTPgammaS.GST-Rac1 and weakly with GDP.Rac1 but not with GST-Cdc42 or GST-RhoA. The N-terminal domain of Sra-1 (1-407 amino acids) is responsible for the interaction with Rac1. Myc-tagged Sra-1 and the deletion mutant capable of interacting with Rac1, but not the mutants unable to bind Rac1, were colocalized with dominant active Rac1(Val-12) and cortical actin filament at the Rac1(Val-12)-induced membrane ruffling area in KB cells. Sra-1 was cosedimented with filamentous actin (F-actin), indicating that Sra-1 directly interacts with F-actin. These results suggest that Sra-1 is a novel and specific target for Rac1 (Kobayashi, 1998).
The Rho-GTPase Rac1 stimulates actin remodelling at the cell periphery by relaying signals to Scar/WAVE proteins leading to activation of Arp2/3-mediated actin polymerization. Scar/WAVE proteins do not interact with Rac1 directly, but instead assemble into multiprotein complexes, which was shown to regulate their activity in vitro. However, little information is available on how these complexes function in vivo. The specifically Rac1-associated protein-1 (Sra-1) and Nck-associated protein 1 (Nap1) interact with WAVE2 and Abi-1 (e3B1) in resting cells or upon Rac activation. Consistently, Sra-1, Nap1, WAVE2 and Abi-1 translocated to the tips of membrane protrusions after microinjection of constitutively active Rac. Moreover, removal of Sra-1 or Nap1 by RNA interference abrogates the formation of Rac-dependent lamellipodia induced by growth factor stimulation or aluminium fluoride treatment. Finally, microinjection of an activated Rac failed to restore lamellipodia protrusion in cells lacking either protein. Thus, Sra-1 and Nap1 are constitutive and essential components of a WAVE2- and Abi-1-containing complex linking Rac to site-directed actin assembly (Steffen, 2004).
SCAR/WAVE functions downstream of Rac signaling Rac is a Rho-family small GTPase that induces the formation of membrane ruffles.
However, it is poorly understood how Rac-induced reorganization of the actin
cytoskeleton, which is essential for ruffle formation, is regulated. A novel Wiskott-Aldrich syndrome protein (WASP)-family protein, WASP
family Verprolin-homologous protein (WAVE: see Drosophila SCAR), has been identified as a regulator of actin
reorganization downstream of Rac. Ectopically expressed WAVE induces the
formation of actin filament clusters that overlap with the expressed WAVE
itself. In this actin clustering, profilin, a monomeric actin-binding protein
that has been suggested to be involved in actin polymerization, has been shown to be
essential. The expression of a dominant-active Rac mutant induces the
translocation of endogenous WAVE from the cytosol to membrane ruffling areas.
Furthermore, the co-expression of a deltaVPH WAVE mutant that cannot induce
actin reorganization specifically suppresses the ruffle formation induced by
Rac, but has no effect on Cdc42-induced actin-microspike formation, a phenomenon
that is also known to be dependent on rapid actin reorganization. The deltaVPH
WAVE also suppresses membrane-ruffling formation induced by platelet-derived growth factor in Swiss 3T3 cells. Taken together, it is concluded that WAVE plays a critical role downstream of Rac in regulating the actin cytoskeleton required for membrane ruffling (Miki, 1998).
Neural Wiskott-Aldrich syndrome protein (N-WASP) functions in several
intracellular events including filopodium formation, vesicle transport and
movement of Shigella frexneri and vaccinia virus, by stimulating rapid actin
polymerization through the Arp2/3 complex. N-WASP is regulated by the direct
binding of Cdc42, which exposes the domain in N-WASP that activates
the Arp2/3 complex. A WASP-related protein, WAVE/Scar (see Drosophila SCAR), functions in Rac-induced
membrane ruffling; however, Rac does not bind directly to WAVE, raising the
question of how WAVE is regulated by Rac. IRSp53, a
substrate for insulin receptor with unknown function, has been demonstrated to be the 'missing link'
between Rac and WAVE. Activated Rac binds to the amino terminus of IRSp53, and
carboxy-terminal Src-homology-3 domain of IRSp53 binds to WAVE to form a
trimolecular complex. From studies of ectopic expression, it was found that IRSp53 is essential for Rac to induce membrane ruffling, probably because it recruits WAVE, which stimulates actin polymerization mediated by the Arp2/3 complex (Miki, 2000).
Rac signalling to actin -- a pathway that is thought to be mediated by the protein Scar/WAVE (WASP (Wiskott-Aldrich syndrome protein)-family verprolin homologous protein) -- has a principal role in cell motility. In an analogous pathway, direct interaction of Cdc42 with the related protein N-WASP stimulates actin polymerization. For the Rac-WAVE pathway, no such direct interaction has been identified. This study reports a mechanism by which Rac and the adapter protein Nck activate actin nucleation through WAVE1. WAVE1 exists in a heterotetrameric complex that includes orthologues of human PIR121 [p53-inducible messenger RNA with a relative molecular mass (Mr) of 140,000], Nap125 (NCK-associated protein with an Mr of 125,000) and HSPC300. Whereas recombinant WAVE1 is constitutively active, the WAVE1 complex is inactive. It is therefore proposed that Rac1 and Nck cause dissociation of the WAVE1 complex, which releases active WAVE1-HSPC300 and leads to actin nucleation (Eden, 2002).
Members of the Rho family of small GTPases, such as Cdc42 and Rac1, and of the Src homology (SH) domain-containing SH2–SH3 adapter protein family, such as NCK, link extracellular signals and actin nucleation through pathways that include the WASP family of proteins and the actin nucleation machinery—the Arp2/3 complex. All WASP family members contain a conserved verprolin-homology, cofilin-homology, acidic (VCA) domain that directly binds and activates the Arp2/3 complex. The Arp2/3 complex, in turn, catalyses the nucleation of actin filaments. To prevent undesirable spontaneous actin nucleation in the absence of input signals, the activity of the WASP proteins is tightly regulated. For example, N-WASP is found predominantly in an autoinhibited conformation in which the carboxy-terminal VCA domain is occluded through interaction with the amino terminus of the protein. When Cdc42 binds to the Cdc42/Rac1 interactive binding (CRIB) domain of N-WASP or when NCK binds to the polyproline region of N-WASP, this autoinhibition is relieved and the VCA domain is unmasked. Phosphatidylinositol(4,5)bisphosphate (PIP2) can further activate N-WASP in cooperation with NCK or Cdc42 by binding to a basic region of N-WASP (Eden, 2002).
The WAVE proteins (WAVE1, WAVE2 and WAVE3 in mammals and orthologues in Drosophila and Dictyostelium are similar in structure to N-WASP. They all have a C-terminal VCA domain, a polyproline region and a basic region. Unlike N-WASP, WAVE proteins do not contain a CRIB domain, and direct binding of WAVE1 to Rac1 has not been detected. But much evidence suggests that WAVE1 functions downstream of Rac1. WAVE1 is translocated from the cytoplasm to membrane ruffles induced by Rac1, and dominant-negative WAVE1 abolishes the formation of Rac1-dependent lamellipodia and Rac1-dependent neurite extensions. The mechanism of regulation of WAVE1 is likely to be fundamentally different from that of N-WASP: whereas N-WASP is autoinhibited, recombinant WAVE1 is constitutively active in stimulating the actin nucleation activity of Arp2/3. Therefore, WAVE1 activity is either inhibited in trans by other cellular regulators or regulated by post-translational modifications (Eden, 2002).
Although WAVE1 had been implicated as the downstream target of Rac1, no regulatory linkage had been found previously. Consequently, the important Rac1-dependent pathway for actin nucleation has not been described. Similarly, although the association of NCK and Rac1 with NAP125 and PIR121 has been observed in several screens, their role as regulators of actin nucleation has not been shown. These results indicate that WAVE1, like N-WASP and WASP, mediates signals from NCK and the Rho GTPases. The activation mechanisms of WAVE1 and N-WASP are very different: N-WASP is autoinhibited, whereas WAVE1 is trans-inhibited. The action of Rac1 and NCK is to disassemble the trans-inhibited WAVE1 complex, which releases the active WAVE1 protein in association with HSPC300. Consistent with this model, Rac1 and WAVE1 do not colocalize in the lamellipodium: WAVE1 is localized at the extreme edge of the lamellipodium, whereas Rac1 is distributed diffusely over the lamellipodium (Eden, 2002).
Although the predominant regulation of WAVE1 activity described here is relief of trans-inhibition, an additional positive regulation by proteins that bind WAVE1 directly in an activator-independent manner is also possible. For example, IRSp53 has been reported to bind WAVE2 directly and enhance activation of Arp2/3 by recombinant WAVE2. Preliminary data show that HSPC300, which remains associated with WAVE1 after activation, may also have a stimulating function on actin polymerization. The activation and dissociation of the WAVE1 complex process releases a subcomplex of NAP125 and PIR121, and this subcomplex may be free to interact with other cellular components. In this way, a Rac1 or NCK signal might potentially coordinate several cellular processes -- similar to pathways that are activated by the alpha- and the gamma-subunits in heterotrimeric G-protein signalling (Eden, 2002).
Cell migration is driven by actin polymerization at the leading edge of lamellipodia, where WASP family verprolin-homologous proteins (WAVEs) activate Arp2/3 complex. When fibroblasts are stimulated with PDGF, formation of peripheral ruffles precedes that of dorsal ruffles in lamellipodia. WAVE2 deficiency impairs peripheral ruffle formation and WAVE1 deficiency impairs dorsal ruffle formation. During directed cell migration in the absence of extracellular matrix (ECM), cells migrate with peripheral ruffles at the leading edge; WAVE2, but not WAVE1, is essential essential for this directed migration. In contrast, both WAVE1 and WAVE2 are essential for invading migration into ECM, suggesting that the leading edge in ECM has characteristics of both ruffles. WAVE1 is colocalized with ECM-degrading enzyme MMP-2 in dorsal ruffles, and WAVE1-, but not WAVE2-, dependent migration requires MMP activity. Thus, WAVE2 is essential for leading edge extension for directed migration in general and WAVE1 is essential in MMP-dependent migration in ECM (Suetsugu, 2003).
Under uniform stimuli that result in random migration, coordination of the formation of peripheral and dorsal ruffles does not seem to be required, and thus the two types of ruffles develop and appear separately. Formation of both types of ruffles appears to be regulated by Rac, because both peripheral and dorsal ruffle formation are suppressed by DN Rac and Wortmannin, a PI-3 kinase inhibitor. Further, PIP3 also accumulates at dorsal ruffles prior to membrane extension. Therefore, activation of Rac through PIP3 binding Rac GEFs including Vav, Sos, and SWAP-70 is thought to occur also in dorsal ruffles. However, constitutive activation of Rac results in only induction of peripheral ruffles. Since WAVE2 is essential in peripheral ruffle formation under PDGF treatment, constitutively active Rac induction of peripheral ruffles is impaired only in WAVE2-deficient cells. This result suggests that WAVE2 is the primary effecter of Rac in formation of ruffles. Consistently, the adaptor molecule IRSp53, which links WAVE2 and Rac, specifically binds to WAVE2, not to WAVE1. In the actin polymerization assay using WAVE2-deficient cell lysate, Rac-induced actin polymerization is impaired in WAVE2-deficient cell lysate, not in wild-type cell lysate, also indicating that WAVE2 is the primary effecter of Rac (Suetsugu, 2003).
Because constitutive activation of Rac does not cause dorsal ruffles, some additional signals are required for dorsal ruffle formation. The regulation of WRP, a RacGAP that binds to WAVE1 and inactivates Rac, might be involved in dorsal ruffles. Activation of c-Abl and recruitment of c-Cbl are involved in dorsal ruffle formation. c-Abl associates with WAVE1. Abi1 also interacts with WAVE1 and is involved in dorsal ruffle formation. These signals may be required for dorsal ruffle formation through unknown regulation of WAVE1 (Suetsugu, 2003).
Rho family GTPases control cell migration and participate in the regulation of cancer metastasis. Invadopodia, associated with invasive tumour cells, are crucial for cellular invasion and metastasis. To study Rac1 GTPase in invadopodia dynamics, a genetically encoded, single-chain Rac1 fluorescence resonance energy (FRET) transfer biosensor was developed. The biosensor shows Rac1 activity exclusion from the core of invadopodia, and higher activity when invadopodia disappear, suggesting that reduced Rac1 activity is necessary for their stability, and Rac1 activation is involved in disassembly. Photoactivating Rac1 at invadopodia confirmed this previously unknown Rac1 function. This study describes an invadopodia disassembly model, where a signalling axis involving TrioGEF, Rac1, Pak1, and phosphorylation of cortactin, causes invadopodia dissolution. This mechanism is critical for the proper turnover of invasive structures during tumour cell invasion, where a balance of proteolytic activity and locomotory protrusions must be carefully coordinated to achieve a maximally invasive phenotype (Moshfegh, 2014).
Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. This study identified a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and has shown that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac-Arpin-Arp2/3 inhibitory circuit with the Rac-WAVE-Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration (Dang, 2013).
Epithelial cell-cell junctions are specialised structures connecting individual cells in epithelial tissues. They are dynamically and functionally linked to the actin cytoskeleton. Disassembly of these junctions is a key event during physiological and pathological processes, but how this influences gene expression is largely uncharacterised. This study investigated whether junction disassembly regulates transcription by serum response factor (SRF) and its coactivator MAL/MRTF. Ca2+-dependent dissociation of epithelial integrity was found to correlate strictly with SRF-mediated transcription. In cells lacking E-cadherin expression, no SRF activation was observed. Direct evidence is provided that signalling occurs via monomeric actin and MAL. Dissociation of epithelial junctions is accompanied by induction of RhoA and Rac1. However, using clostridial cytotoxins, it was demonstrated that Rac, but not RhoA, is required for SRF and target gene induction in epithelial cells, in contrast to serum-stimulated fibroblasts. Actomyosin contractility is a prerequisite for signalling but failed to induce SRF activation, excluding a sufficient role of the Rho-ROCK-actomyosin pathway. It is concluded that E-cadherin-dependent cell-cell junctions facilitate transcriptional activation via Rac, G-actin, MAL and SRF upon epithelial disintegration (Busche, 2008).
The SWI/SNF chromatin remodelling complexes are important regulators of transcription; they consist of large multisubunit assemblies containing either Brm or Brg1 as the catalytic ATPase subunit and a variable subset of approximately 10 Brg/Brm-associated factors (BAF). Among these factors, BAF60 proteins (BAF60a, BAF60b or BAF60c), which are found in most complexes, are thought to bridge interactions between transcription factors and SWI/SNF complexes. This study reports on a Rac-dependent process leading to BAF60b ubiquitination. Using two-hybrid cloning procedures, this study identified a mammalian RING finger protein homologous to Drosophila Unkempt as a new partner of the activated form of RacGTPases; mammalian Unkempt specifically binds to BAF60b and promotes its ubiquitination in a Rac1-dependent manner. Immunofluorescence studies demonstrated that Unkempt is primarily localized in the cytoplasmic compartment, but has the ability to shuttle between the nucleus and the cytoplasm, suggesting that the Rac- and Unkempt-dependent process leading to BAF60b ubiquitination takes place in the nuclear compartment. Ubiquitinated forms of BAF60b were found to accumulate upon treatment with the proteasome inhibitor MG132, indicating that BAF60b ubiquitination is of the degradative type and could regulate the level of BAF60b in SWI/SNF complexes. These observations support the new idea of a direct connection between Rac signalling and chromatin remodelling (Lores, 2010).
Although the results reported above are consistent with BAF60b being ubiquitinated through a Rac- and Unkempt-dependent process, the molecular composition of the E3 ligase involved and the role of Unkempt RING finger remain uncertain. On the basis of the results of a mutational analysis, it appears that the RING finger of exogenously expressed Unkempt is not critically involved in the ubiquitination reaction. A possible explanation is that exogenously expressed mutants of Unkempt form dimers/oligomers with endogenous Unkempt and/or associates with other RING finger protein(s), resulting in active E3 ligase. As already mentioned, there are multiple examples of RING E3s, the activity of which critically depends on multiprotein complexes, including homo- or hetero-oligomers of RING finger proteins. Of note, interaction between RING finger proteins does not necessarily depend on the RING finger motif itself. Thus, yBRE1, a RING finger protein involved in H2B ubiquitination in budding yeast, forms a homomeric complex, possibly a tetramer, through multiple intermolecular interactions, implicating only minimally the C-terminal RING finger. Similarly, in human, the RING finger type paralogs hBRE1A and hBRE1B form a heterotetramer and are both required for H2B ubiquitination, but the hBRE1B RING finger is dispensable. Another interesting example is provided by Pirh2, a p53-induced RING finger E3 ligase promoting ubiquitination and degradation of p53; very recently, isoforms of Pirh2 with a disrupted RING finger motif have been found capable of promoting p53 ubiquitination, possibly through their ability to interact directly with MDM2, the principal E3 ligase for p53. The RING finger protein Unkempt may share similarities with these models. It was recently observed that UNK-C-ter is capable of forming homomeric complexes in GST pull-down experiments; however, it remains to be demonstrated that an E3 ligase activity is associated with Unkempt homomers (or with heteromers involving an unidentified RING finger protein) and whether and how RacGTP regulates this putative E3 ligase. To address these issues, in vitro studies aimed at analysing intrinsic E3 ligase activity of recombinant Unkempt will be required (Lores, 2010).
The results also raise the questions of the physiological relevance and significance of BAF60b ubiquitination. Unfortunately, using available antibodies to BAF60b, no ubiquitinated forms of endogenous BAF60b were detected. However, in HeLa cells expressing exogenous BAF60b, it was found that BAF60b is significantly ubiquitinated, even in the absence of exogenous Unkempt; in addition, the ubiquitinated forms of BAF60b strongly accumulated in the presence of MG132, suggesting that the fate of ubiquitinated BAF60b is proteasomal degradation. Thus, it may be that ubiquitination results in degradation of an excess of BAF60b subunits, thereby allowing the stoichiometry of SWI/SNF complexes to be maintained. Another interesting possibility would be that BAF60b, alone or in complex with Unkempt, interacts with other unidentified substrates of Unkempt-dependent E3 ligase. As previously mentioned, BAF60 proteins are thought to bridge interactions between transcription factors and SWI/SNF complexes; therefore, candidate substrates include other constituents of SWI/SNF complexes, some of which have been found to be regulated by proteasomal degradation, and transcription factors targeted by BAF60b that remain to be defined (Lores, 2010).
Whatever the precise mechanisms are, Unkempt may be importantly linked to the physiological control of the SWI/SNF complexes, thus opening up a direct connection between Rac signalling and chromatin remodelling (Lores, 2010).
Rac is a small GTPase of the Rho family that mediates stimulus-induced actin cytoskeletal reorganization to generate
lamellipodia. Little is known about the signaling pathways that link Rac activation to changes in actin filament dynamics.
Cofilin is known to be a potent regulator of actin filament dynamics, and its ability to bind and depolymerize actin is abolished
by phosphorylation of the serine residue at 3; however, the kinases responsible for this phosphorylation have not been identified.
LIM-kinase 1 (LIMK-1), a serine/threonine kinase containing LIM and PDZ domains, phosphorylates
cofilin at Ser 3, both in vitro and in vivo. When expressed in cultured cells, LIMK-1 induces actin reorganization and reverses
cofilin-induced actin depolymerization. Expression of an inactive form of LIMK-1 suppresses lamellipodium formation
induced by Rac or insulin. Insulin and an active form of Rac increase the activity of LIMK-1. Taken together,
these results indicate that LIMK-1 participates in Rac-mediated actin cytoskeletal reorganization, probably by phosphorylating
cofilin (Yang, 1998).
Small GTPases of the Rho family regulate signaling pathways that control actin cytoskeletal structures. In Swiss 3T3 cells, RhoA
activation leads to stress fiber and focal adhesion formation; Rac1 activation to lamellipoda and membrane ruffles, and Cdc42 to microspikes
and filopodia. Recently identified have been several downstream molecules mediating these effects. Evidence is provided that the intracellular localization of the actin binding protein cortactin, a Src kinase substrate, is regulated by the activation
of Rac1. Cortactin redistributes from the cytoplasm into membrane ruffles as a result of growth factor-induced Rac1 activation, and
this translocation is blocked by expression of dominant negative Rac1N17. Expression of constitutively active Rac1L61 evokes the
translocation of cortactin from cytoplasmic pools into peripheral membrane ruffles. Expression of mutant forms of the
serine/threonine kinase PAK1, a downstream effector of Rac1 and Cdc42 that has recently been demonstrated to trigger cortical actin
polymerization and membrane ruffling, also leads to the translocation of cortactin to the cell cortex, although this is effectively
blocked by coexpression of Rac1N17. Collectively these data provide evidence for cortactin as a putative target of Rac1-induced
signal transduction events involved in membrane ruffling and lamellipodia formation (Weed, 1998).
Cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells depend directly on
the regulated dynamics of the actin cytoskeleton. To understand the mechanisms of these basic cellular processes, the
signaling pathways that link external signals to the regulation of the actin cytoskeleton need to be characterized. A pathway has been identified for the regulation of cofilin, a ubiquitous actin-binding protein that is essential for effective
depolymerization of actin filaments. In vivo, cofilin has been shown to be essential for cytokinesis, endocytosis and other cell processes that require rapid turnover of actin filaments. In vitro, cofilin binds to both actin monomers and polymers, and promotes the disassembly of actin filaments. Cofilin is regulated by phosphorylation of the serine residue at position 3, which inhibits its actin-binding and depolymerization activities. Stimuli that induce the production of lamellipodia relieve this inhibition by causing the rapid dephosphorylation of cofilin. LIM-kinase 1, also known as KIZ, is a protein kinase with two amino-terminal LIM motifs that induces stabilization of F-actin structures in transfected cells. Dominant-negative LIM-kinasel inhibits the accumulation of the F-actin. Phosphorylation experiments in vivo and in vitro provide evidence that cofilin is a physiological substrate of LIM-kinase 1. Phosphorylation by LIM-kinase 1 inactivates cofilin, leading to accumulation of actin filaments. Constitutively active Rac augments cofilin phosphorylation and LIM-kinase 1 autophosphorylation, whereas phorbol ester inhibits these processes. These results define a mechanism for the regulation of cofilin and hence of actin dynamics in vivo. By modulating the stability of actin cytoskeletal structures, this pathway should play a central role in regulating cell motility and morphogenesis (Arber, 1998).
The currently known members of the MAP kinase family
include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun
N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP
kinases. Overexpression of p21-activated
kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a
lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can
stimulate the activity of each of these MAP kinases. Although neither activated Rac
nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative
Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation
of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway.
Constitutively active Rac2, Cdc42hs, and RhoA synergize with an
activated form of Raf to increase ERK2 activity. These findings reveal a previously
unrecognized connection between Rho family small G proteins and the ERK pathway (Frost, 1996).
Among the mechanisms by which the Ras oncogene induces cellular transformation, Ras activates the mitogen-activated protein kinase (MAPK or ERK) cascade and a related cascade leading to activation of Jun kinase (JNK or SAPK). JNK is
additionally regulated by the Ras-related G proteins Rac and Cdc42. Ras also regulates the actin cytoskeleton through an incompletely elucidated Rac-dependent mechanism. A candidate for the physiological effector for both JNK and actin
regulation by Rac and Cdc42 is the serine/threonine kinase Pak (p65pak). Expression of a catalytically inactive mutant Pak, Pak1(R299), inhibits Ras transformation of Rat-1 fibroblasts but not of NIH 3T3 cells. Typically, 90 to 95%
fewer transformed colonies are observed in cotransfection assays with Rat-1 cells. Pak1(R299) does not inhibit transformation by the Raf oncogene, indicating that
inhibition is specific for Ras. Rat-1 cell lines expressing Pak1(R299) are highly resistant to Ras transformation, while cells expressing wild-type Pak1 are efficiently transformed by Ras. Pak1(L83,L86,R299), a mutant that fails to bind
either Rac or Cdc42, also inhibits Ras transformation. Rac and Ras activation of JNK is inhibited by Pak1(R299) but not by Pak1(L83,L86,R299). Ras activation of ERK is inhibited by both Pak1(R299) and Pak1(L83,L86,R299), while neither
mutant inhibits Raf activation of ERK. These results suggest that Pak1 interacts with
components essential for Ras transformation and that inhibition can be uncoupled from
JNK but not ERK signaling (Tang, 1997).
The c-jun proto-oncogene encodes a transcription factor that is activated by mitogens both
transcriptionally and as a result of phosphorylation by Jun N-terminal kinase (JNK). The
cellular signaling pathways involved in epidermal growth factor (EGF) induction of the c-jun promoter have been investigated.
Two sequence elements that bind ATF1 (a leucine zipper DNA binding protein) and MEF2D transcription factors are required
in HeLa cells, although these elements are not sufficient for maximal induction. Activated forms of Ras, RacI,
Cdc42Hs, and MEKK increase expression of the c-jun promoter, while dominant negative forms of
Ras, RacI, and MEK kinase (MEKK) inhibit EGF induction. These results
suggest that EGF activates the c-jun promoter by a Ras-to-Rac-to-MEKK pathway. No change is found in protein binding to the jun ATF1 site in EGF-treated cells. A potential mechanism for regulation of ATF1 and CREB is phosphorylation (Clarke, 1997).
The protein product of the human vav oncogene (Vav) exhibits a number of structural motifs suggestive of a role in signal transduction pathways, including a leucine-rich region, a plekstrin homology (PH) domain, a cysteine-rich domain, two SH3 regions, an SH2 domain, and a central Dbl homology (DH) domain. However, the transforming pathway(s) activated by Vav has not yet been elucidated. Interestingly, DH domains are frequently found in guanine nucleotide-exchange factors for small GTP-binding proteins of the Ras and Rho families (Crespo, 1996).
Are either MAPK or JNK downstream components of the Vav signaling
pathways?This question was prompted by the
structural similarity between Vav and other guanine nucleotide exchange factors for
small GTP-binding proteins, together with the recent identification of biochemical
routes specific for members of the Ras and Rho family of GTPases. Neither Vav nor the product of the vav proto-oncogene, proto-Vav, can enhance the
enzymatic activity of MAPK. While proto-Vav can slightly elevate JNK/SAPK activity,
oncogenic Vav potently activates JNK/SAPK to an extent comparable to that elicited
by two guanine-nucleotide exchange factors for Rho family members Dbl and Ost. Point mutations in conserved residues within the cysteine rich and
DH domains of Vav both prevent Vav's ability to activate JNK/SAPK and render Vav
oncogenically inactive. Coexpression of the Rac-1 N17
dominant inhibitory mutant dramatically diminishes JNK/SAPK stimulation by Vav, as well as reduces the focus-forming ability of Vav in NIH3T3 murine fibroblasts. Taken together, these findings provide the first evidence that Rac-1 and JNK are integral components of the Vav signaling pathway (Crespo, 1996).
The Rho family of small GTPases includes critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to the cell nucleus. Other family members are the JNK/SAPK signaling pathway, the c-fos serum response factor, and the p70 S6 kinase. A novel signaling pathway is activated by the Rho proteins. This pathway may be responsible for biological activities carried out by Rho proteins, including cytoskeleton organization,
transformation, apoptosis, and metastasis. The human RhoA, CDC42, and Rac-1
proteins efficiently induce the transcriptional activity of nuclear factor KB (NF-KB) by a mechanism that involves phosphorylation of IKappaBalpha and translocation of p50/p50 and p50/p65 dimers to the nucleus, independent of the involvement of Ras GTPase and the Raf-1 kinase. Activation of NF-KB by TNFalpha depends on CDC42 and RhoA because this activity is drastically inhibited by CDC42 and RhoA dominant-negative mutants. In contrast, activation of NF-KB by UV light
is not affected by Rho, CDC42, or Rac-1 dominant-negative mutants. Thus,
members of the Rho family of GTPases are involved specifically in the regulation of NF-KB-dependent transcription (Perona, 1997).
The c-fos serum response element (SRE) forms a ternary complex with the
transcription factors SRF (serum response factor) and TCF (ternary complex factor).
By itself, SRF can mediate transcriptional activation induced by serum,
lysophosphatidic acid, or intracellular activation of heterotrimeric G proteins. Activated
forms of the Rho family GTPases RhoA, Rac1, and CDC42Hs also activate
transcription via SRF and act synergistically at the SRE with signals that activate
TCF. Functional Rho is required for signaling to SRF by several stimuli, but not by
activated CDC42Hs or Rac1. Activation of the SRF-linked signaling pathway does not
correlate with activation of the MAP kinases ERK, SAPK/JNK, or MPK2/p38.
Functional Rho is required for regulated activity of the c-fos promoter. These results
establish SRF as a nuclear target of a novel Rho-mediated signaling pathway (Hill, 1995).
Kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in
Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. Genetic and biochemical studies
suggest that KSR is a positive regulator of Ras signaling that functions between Ras and Raf or in a pathway parallel to
Raf. The effect of mammalian KSR expression was examined on the activation of extracellular ligand-regulated (ERK)
mitogen-activated protein (MAP) kinase in fibroblasts. Ectopic expression of KSR inhibits the activation of ERK MAP
kinase by insulin, phorbol ester, or activated alleles of Ras, Raf, and mitogen and extracellular-regulated kinase.
Expression of deletion mutants of KSR demonstrates that the KSR kinase domain is necessary and sufficient for the
inhibitory effect of KSR on ERK MAP kinase activity. KSR inhibits cell transformation by activated RasVal-12 but has
no effect on the ability of RasVal-12 to induce membrane ruffling. These data indicate that KSR is a potent modulator of a
signaling pathway essential to normal and oncogenic cell growth and development (Joneson, 1998).
The Rho, Rac and Cdc42 GTPases coordinately regulate the organization of the actin cytoskeleton and the JNK MAP kinase pathway. Mutational analysis of Rac has shown that these two activities are mediated by distinct cellular targets, though their identities are not known. Two Rac targets, p65(PAK) and MLK, are ser/thr kinases that have been reported to be capable of activating the JNK pathway. Evidence is presented that neither is the Rac target mediating JNK activation in Cos-1 cells. Yeast two-hybrid selection identified POSH as a new target of Rac. This protein consists of four SH3 domains and ectopic expression leads to the activation of the JNK pathway and to nuclear translocation of NF-kappaB. When overexpressed in fibroblasts, POSH is a strong inducer of apoptosis. It is proposed that POSH acts as a scaffold protein and contributes to Rac-induced signal transduction pathways leading to diverse gene transcriptional changes (Tapon, 1998).
The multidomain protein POSH (plenty of SH3s) acts as a scaffold for the JNK pathway of neuronal death. This pathway consists of a sequential cascade involving activated Rac1/Cdc42, mixed-lineage kinases (MLKs), MAP kinase kinases (MKKs) 4 and 7, c-Jun N-terminal kinases (JNKs) and c-Jun, and is required for neuronal death induced by various means including nerve growth factor (NGF) deprivation. In addition to binding GTP-Rac1, POSH binds MLKs both in vivo and in vitro, and complexes with MKKs 4 and 7 and with JNKs. POSH overexpression promotes apoptotic neuronal death and this is suppressed by dominant-negative forms of MLKs, MKK4/7 and c-Jun, and by an MLK inhibitor. Moreover, a POSH antisense oligonucleotide and a POSH small interfering RNA (siRNA) suppress c-Jun phosphorylation and neuronal apoptosis induced by NGF withdrawal. Thus, POSH appears to function as a scaffold in a multiprotein complex that links activated Rac1 and downstream elements of the JNK apoptotic cascade (Xu, 2003).
Actin polymerization is essential for a variety of cellular processes including movement, cell division and shape change. The induction of actin polymerization requires the generation of free actin filament barbed ends, which results from the severing or uncapping of pre-existing actin filaments, or de novo nucleation, initiated by the Arp2/3 complex. Although little is known about the signaling pathways that regulate actin assembly, small GTPases of the Rho family appear to be necessary. In thrombin-stimulated platelets, the Rho family GTPase Rac1 induces actin polymerization by stimulating the uncapping of actin filament barbed ends. The mechanism by which Rac regulates uncapping is unclear, however. Rac interacts with a type Ialpha phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) in a GTP-independent manner. Because PIP 5-kinases synthesize phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], a lipid that dissociates capping proteins from the barbed ends of actin filaments, they are good candidates for mediating the effects of Rac on actin assembly. Here, the Rac-associated PIP 5-kinase has been idenfied as the PIP 5-kinase isoforms alpha and beta. When added to permeabilized platelets, PIP 5-kinase alpha induces actin filament uncapping and assembly. In contrast, a kinase-inactive PIP 5-kinase alpha mutant fails to induce actin assembly and blocks assembly stimulated by thrombin or Rac. Furthermore, thrombin- or Rac-induced actin polymerization is inhibited by a point mutation in the carboxyl terminus of Rac that disrupts PIP 5-kinase binding. These results demonstrate that PIP 5-kinase alpha is a critical mediator of thrombin- and Rac-dependent actin assembly (Tolias, 2000).
Mutations in the neurofibromatosis type II (NF2) tumor suppressor predispose humans and mice to tumor development. The study of Nf2+/- mice has demonstrated an additional effect of Nf2 loss on tumor metastasis.
The NF2-encoded protein, merlin, belongs to the ERM (ezrin, radixin, and moesin) family of cytoskeleton-membrane linkers. However, the molecular basis for the tumor- and metastasis-suppressing activity of merlin is unknown. This study places merlin in a signaling pathway downstream of the small GTPase Rac. Expression of activated Rac induces phosphorylation and decreased association of merlin with the cytoskeleton. Furthermore, merlin overexpression inhibits Rac-induced signaling in a phosphorylation-dependent manner. Finally, Nf2-/- cells exhibit characteristics of cells expressing activated alleles of Rac. These studies provide insight into the normal cellular function of merlin and how Nf2 mutation contributes to tumor initiation and progression (Shaw, 2001).
The results indicate that merlin is regulated by Rac. Although phosphopeptide mapping experiments indicate that merlin phosphorylation is complex, the existing data suggest that Rac-induced phosphorylation inactivates merlin. Phosphorylation of the Rac-responsive S518 residue inhibits merlin self-association, weakens its association with the cytoskeleton, and compromises its ability to inhibit
Rac-mediated signaling. This suggests that the closed or oligomerized form of merlin is the active, growth suppressing form. This is consistent with the observations that the same
amino acid residues necessary for self-association are also required for the growth- and motility- suppressing function of merlin in schwannoma cells. Importantly, Rac is activated by growth factor stimulation and matrix adhesion, conditions which also stimulate endogenous merlin
phosphorylation. The finding that merlin is
phosphorylated in response to activation of Rac/Cdc42 and not Rho would indicate that merlin and the ERMs
are regulated in distinct ways. Together with the observation that merlin can heterodimerize with ERM proteins, this suggests that this family of proteins could serve to coordinate the activities of these GTPases (Shaw, 2001 and references therein.
The observation that merlin is phosphorylated in response to Rac activation might have suggested that merlin is an effector of Rac and thus required for some downstream functions of Rac. Instead, the results are consistent
with a model wherein merlin normally acts to attenuate Rac signaling: (1) overexpression of merlin inhibits
JNK activity as well as Rac-induced AP-1 activity and transformation; (2) Nf2-deficient cells exhibit
phenotypes that are consistent with hyperactivation of Rac signaling including increased activation of JNK,
AP-1, membrane ruffling, and motility. Notably, overexpression of merlin
inhibits cell spreading and motility. Together these observations are consistent with a model in which merlin functions as a sensor of Rac signaling. Phosphorylation of merlin in response to Rac activation or other stimuli inactivates merlin, thereby potentiating Rac signaling; dephosphorylation would restore its inhibitory function (Shaw, 2001 and references therein).
Studies of ion channel regulation by G proteins have focused on the larger, heterotrimeric GTPases, which are activated by heptahelical membrane receptors. In contrast, studies of the Rho family of smaller, monomeric, Ras-related GTPases, which are activated by cytoplasmic guanine nucleotide exchange factors, have focused on their role in cytoskeletal regulation. This study demonstrates novel functions for the Rho family GTPases Rac and Rho in the opposing hormonal regulation of voltage-activated, ether-a-go-go-related potassium channels (ERG) in a rat pituitary cell line, GH4C1. The hypothalamic neuropeptide, thyrotropin-releasing hormone (TRH) inhibits ERG channel activity
through a PKC-independent process that is blocked by RhoA(19N) and the Clostridium botulinum C3 toxin, which inhibit Rho signaling. The constitutively active, GTPase-deficient mutant of RhoA(63L) rapidly inhibits the channels when the protein is dialysed directly into the cell through the patch pipette, and inhibition persists when the protein is overexpressed. In contrast, GTPase-deficient Rac1(61L) stimulates ERG channel activity. The thyroid hormone triiodothyronine (T3), which antagonizes TRH action in the pituitary, also stimulates ERG channel activity through a rapid process that is blocked by Rac1(17N) and wortmannin but not by RhoA(19N). It is concluded that Rho stimulation by G13-coupled receptors and Rac stimulation by nuclear hormones through PI3-kinase may be general mechanisms for regulating ion channel activity in many cell types. Disruption of these novel signaling cascades is predicted to contribute to several specific human neurological diseases, including epilepsy and deafness (Storey, 2002).
During cytokinesis, regulatory signals are presumed to emanate from the mitotic spindle. However, what these signals are and how they lead to the spatiotemporal changes in the cortex structure, mechanics, and regional contractility are not well understood in any system. To investigate pathways that link the microtubule network to the cortical changes that promote cytokinesis, chemical genetics was used in Dictyostelium to identify genetic suppressors of nocodazole, a microtubule depolymerizer. 14-3-3 is enriched in the cortex, helps maintain steady-state microtubule length, contributes to normal cortical tension, modulates actin wave formation, and controls the symmetry and kinetics of cleavage furrow contractility during cytokinesis. Furthermore, 14-3-3 acts downstream of a Rac small GTPase (RacE), associates with myosin II heavy chain, and is needed to promote myosin II bipolar thick filament remodeling. It is concluded that 14-3-3 connects microtubules, Rac, and myosin II to control several aspects of cortical dynamics, mechanics, and cytokinesis cell shape change. Furthermore, 14-3-3 interacts directly with myosin II heavy chain to promote bipolar thick filament remodeling and distribution. Overall, 14-3-3 appears to integrate several critical cytoskeletal elements that drive two important processes-cytokinesis cell shape change and cell mechanics (Zhou, 2010).
Rac involvement in adhesion, cell spreading, and cell migration Cadherins (See Drosophila Shotgun) are calcium-dependent cell-cell adhesion molecules that require the interaction of the
cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship
between cadherin receptors and actin filament organization, whether or not members of the
Rho family of small GTPases are necessary for cadherin adhesion was investigated. In fibroblasts, the Rho family
members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia,
respectively. In epithelial cells, Rho and Rac are required for the establishment of
cadherin-mediated cell-cell adhesion and the actin reorganization necessary to stabilize the receptors at
sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removes cadherin
complexes from junctions induced for up to 3 h, while desmosomes are not perturbed. In addition,
withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and
integrins, both microfilament-associated receptors. The concerted action of Rho
and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac is not
sufficient to stabilize cadherin dependent cell-cell contacts when endogenous Rho is inhibited. Upon
induction of calcium-dependent intercellular adhesion, there is a rapid accumulation of actin at sites
of cell-cell contacts, which is prevented by blocking cadherin function, Rho or Rac activity.
However, if cadherin complexes are clustered by specific antibodies attached to beads, actin
recruitment to the receptors is perturbed by inhibiting Rac but not Rho. These results provide new
insights into the role of the small GTPases in the cadherin-dependent cell-cell contact formation and
the remodeling of actin filaments in epithelial cells (Braga, 1997).
Leukocyte adhesion to the extracellular matrix (ECM) is tightly controlled and is vital for the immune response. Circulating lymphocytes leave the bloodstream and adhere to ECM components at sites of inflammation and lymphoid tissues. Mechanisms for regulating T-lymphocyte-ECM adhesion include (1) an alteration in the affinity of cell surface integrin receptors for their extracellular ligands and (2) an alteration of events following postreceptor occupancy (e.g., cell spreading). Whereas H-Ras and R-Ras affect T-cell adhesion by altering the affinity state of the integrin receptors, no signaling molecule has been identified for the second mechanism. Expression of an activated mutant of Rac triggers dramatic spreading of T cells and their increased adhesion on immobilized fibronectin in an integrin-dependent manner. This effect is not mimicked by expression of activated mutant forms of Rho, Cdc42, H-Ras, or ARF6, indicating the unique role of Rac in this event. The Rac-induced spreading is accompanied by specific cytoskeletal rearrangements. Also, a clustering of integrins at sites of cell adhesion and at the peripheral edges of spread cells is observed. Expression of RacV12 does not alter the level of expression of cell surface integrins or the affinity state of the integrin receptors. Moreover, Rac plays a role in the regulation of T-cell adhesion by a mechanism involving cell spreading, rather than by altering the level of expression or the affinity of the integrin receptors. The Rac-mediated signaling pathway leading to spreading of T lymphocytes does not require activation of c-Jun kinase, serum response factor, or pp70(S6 kinase) but appears to involve a phospholipid kinase (D'Souza-Schorey, 1998).
Adhesion to ECM is required for many cell functions including cytoskeletal organization, migration, and proliferation. When cells first adhere to extracellular matrix, they spread rapidly by extending filopodia-like projections and
lamellipodia. These structures are similar to the Rac- and Cdc42-dependent structures observed in growth factor-stimulated
cells. The involvement of Rac and Cdc42 in adhesion and spreading on the ECM protein fibronectin was investigated. Integrin-dependent adhesion leads to the rapid activation of p21-activated kinase, a downstream effector of Cdc42
and Rac, suggesting that integrins activate at least one of these GTPases. Dominant negative mutants of Rac and Cdc42 inhibit
cell spreading in such a way as to suggest that integrins activate Cdc42, which leads to the subsequent activation of Rac; both
GTPases then contribute to cell spreading. These results demonstrate that initial integrin-dependent activation of Rac and
Cdc42 mediates cell spreading (Price, 1998).
The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the
Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, an examination was made of the
roles of Cdc42-, Rac-, and Rho-dependent signaling pathways in the regulation of cytoskeleton during integrin-mediated adhesion and
cell spreading, using the dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix
protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation,
some phosphorylation is observed of the focal adhesion kinase (FAK) and Src, which occurs independent of Rho family GTPases.
However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Rho
dependence is found for the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also
stimulates membrane ruffling; this ruffling is independent of Rho but is dependent on both Cdc42 and Rac.
Cdc42 controls the integrin-dependent activation of extracellular signal-regulated kinase 2 and of
Akt, a kinase whose activity is dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent
membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that
regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42,
Rac, and Rho, all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture.
Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control
adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior (Clark, 1998).
Growth factors promote cell survival and cell motility, presumably through the activation of Akt and the Rac and Cdc42 GTPases, respectively. Because Akt is dispensable for Rac/Cdc42 regulation of actin reorganization, it has been assumed that Rac and Cdc42 stimulate cell motility independent of Akt in mammalian
cells. However, this study demonstrates that Akt is essential for Rac/Cdc42-regulated cell motility in mammalian fibroblasts. A dominant-negative Akt inhibits cell motility stimulated by Rac/Cdc42 or by PDGF treatment, without affecting ruffling membrane-type actin reorganization. Akt is activated by expression of Rac and Cdc42; colocalization of endogenous phosphorylated Akt with Rac and Cdc42 is observed at the leading edge of fibroblasts. Importantly, expression of active Akt
but not the closely related kinase SGK is sufficient for increasing cell motility. This effect of Akt is cell autonomous and not mediated by inhibition of GSK3. Dominant-negative Akt but not SGK reverses the increased cell motility phenotype of fibroblasts lacking the PTEN tumor suppressor gene. Taken
together, these results suggest that Akt promotes cell motility downstream of Rac/Cdc42 in growth factor-stimulated cells and in invasive PTEN-deficient cells (Higuchi, 2002).
GTPases of the Rho family regulate actinomyosin-based
contraction in non-muscle cells. Activation of Rho
increases contractility, leading to cell rounding and neurite
retraction in neuronal cell lines. Activation of Rac promotes
cell spreading and interferes with Rho-mediated cell
rounding. Activation of Rac may
antagonize Rho by regulating phosphorylation of the
myosin-II heavy chain. Stimulation of PC12 cells or N1E-115
neuroblastoma cells with bradykinin induces
phosphorylation of threonine residues in the myosin-II
heavy chain; this phosphorylation is Ca2+ dependent and
regulated by Rac. Both bradykinin-mediated and
constitutive activation of Rac promote cell spreading,
accompanied by a loss of cortical myosin II. These results
identify the myosin-II heavy chain as a new target of
Rac-regulated kinase pathways, and implicate Rac as a
Rho antagonist during myosin-II-dependent cell-shape
changes (van Leeuwen, 1999).
The molecular events responsible for
Rac-mediated cytoskeletal changes are not well understood, but they
involve activation of serine/threonine-kinase pathways. To search for
stimuli that induce Rac-dependent changes in neuronal cell lines, an immobilized fusion protein consisting of glutathione-S-transferase fused
to Pak1 was used to measure activation of Rac in lysates of rat PC12 cells in
response to several receptor agonists. The serine/threonine kinase Pak1 is
a downstream effector of both Rac and Cdc42 that specifically binds these
GTPases in their active (GTP-bound) states. Stimulation of PC12 cells
with the neuropeptide bradykinin leads to activation of Rac without inducing
a measurable change in Cdc42 activity. Ectopic expression of constitutively active Tiam1 (C1199Tiam1), a guanine-nucleotide-exchange factor for Rac, potently activates Rac and also increases the amount of GTP-bound Cdc42 to some extent in these cells (van Leeuwen, 1999).
Pak serine/threonine kinases are
activated directly by GTP-bound Rac or Cdc42 and are thought to be
important in the regulation of the actinomyosin cytoskeleton downstream of
these GTPases. To investigate a role for this kinase family in MHC
phosphorylation, dominant-negative Pak1(L83, L86,
R299) was overexpressed in PC12 cells. This kinase-defective mutant no longer binds Rac or
Cdc42, thus avoiding complicating effects resulting from titration
of GTP-bound Rac or Cdc42. Similar to dominant-negative RacN17, Pak1(L83, L86, R299) interfers with bradykinin-induced MHC
phosphorylation. Most cells expressing dominant-negative
Pak1(L83, L86, R299) are round and do not spread in response to
bradykinin. Moreover, myosin II in these cells remains
associated with F-actin at the cell cortex even after stimulation with
bradykinin (van Leeuwen, 1999).
The morphological consequences of Pak1 activation were investigated. Overexpression of either wild-type Pak1 or
Pak1(E423), an activated variant of this kinase, induces cell spreading
accompanied by some redistribution of myosin II, although the observed
changes are very different from those produced by C1199Tiam1 or
Rac1V12. The prominent lamellae, which do not appear to
contain any myosin II, as observed in Tiam1- and RacV12-expressing
cells, are not seen in the Pak1-overexpressing cells. Whereas
dominant-negative Pak1 clearly inhibits bradykinin-induced MHC
phosphorylation, overexpression of wild-type Pak1 or Pak1(E423) is
not sufficient to promote MHC phosphorylation, in either the presence or
the absence of bradykinin. It is speculated that these
differences between the results of Rac and Pak activation either reflect
improper localization of the overexpressed kinase or otherwise indicate
that another member of this kinase family, and not Pak1, is involved in
regulating MHC phosphorylation. A similar discrepancy was found in
Rat-1 fibroblasts, where dominant-negative Pak1(L83, L86, R299)
inhibits Ras transformation, whereas an activated kinase does not
cooperate with either Ras or Raf in cell transformation. Indeed, instead of
Pak1, Pak3 has been shown to activate Raf-1 downstream of Ras17.
Together, these results indicate that a Pak-like kinase activity may regulate
MHC phosphorylation and cell spreading. The identity of the kinase
involved remains to be established (van Leeuwen, 1999).
MHC phosphorylation is Ca2+ dependent. Apparently, bradykinin, which signals through G-protein-coupled receptors, provides extra signals to increase MHC
phosphorylation. Using pharmacological inhibitors, a determination was made of which
bradykinin-induced second-messenger pathways cooperate with Rac to
induce MHC phosphorylation. MHC phosphorylation is completely dependent on the influx of extracellular Ca2+. Chelation of Ca2+ from the tissue-culture medium with
EGTA abolishes phosphorylation in response to bradykinin.
MHC phosphorylation is also effectively blocked by the presence of an inhibitor of receptor-operated Ca2+-channels. Even the sustained increase in MHC
phosphorylation observed in cells overexpressing Tiam1 is reduced to
undetectable levels in cells that have been depleted of Ca2+ by pretreatment
with a membrane-permeable Ca2+ chelator BAPTA-AM.
Conversely, Ca2+ influx, artificially induced with the Ca2+ ionophore
ionomycin, is sufficient to induce phosphorylation of the MHC.
These results indicate that threonine phosphorylation of the myosin-II
heavy chain involves a calcium-dependent kinase pathway, which appears
to be regulated or sensitized by Rac (van Leeuwen, 1999).
The establishment of cadherin-dependent cell-cell contacts in human epidermal keratinocytes are known to be regulated by the
Rac1 small GTP-binding protein, although the mechanisms by which Rac1 participates in the assembly or disruption of cell-cell
adhesion are not well understood. Green fluorescent protein (GFP)-tagged Rac1 expression vectors were used to examine the subcellular distribution of Rac1 and its effects on E-cadherin-mediated cell-cell adhesion. Microinjection of
keratinocytes with constitutively active Rac1 results in cell spreading and disruption of cell-cell contacts. The ability of active Rac1 to
disrupt cell-cell adhesion is dependent on colony size, with large established colonies being resistant to the effects of active Rac1. Disruption of cell-cell contacts in small preconfluent colonies is achieved through the selective recruitment of E-cadherin-catenin complexes to the perimeter of multiple large intracellular vesicles, which are bounded by GFP-tagged constitutively active Rac1. Similar vesicles were observed in noninjected keratinocytes when cell-cell adhesion is disrupted by removal of extracellular calcium or with the use of an E-cadherin blocking antibody. Moreover, formation of these structures in noninjected keratinocytes is dependent on endogenous Rac1 activity. Expression of GFP-tagged effector mutants of Rac1 in keratinocytes demonstrates that reorganization of the actin cytoskeleton is important for vesicle formation. Characterization of these Rac1-induced vesicles revealsthat they are endosomal in nature and tightly colocalized with the transferrin receptor, a marker for recycling endosomes. Expression of GFP-L61Rac1 inhibits uptake of transferrin-biotin, suggesting that the endocytosis of E-cadherin is a clathrin-independent mechanism. This is supported by the observation that caveolin, but not clathrin, localizes around these structures. Furthermore, an inhibitory form of dynamin, known to inhibit internalization of caveolae, inhibits formation of cadherin vesicles. These data suggest that Rac1 regulates adherens junctions via clathrin independent endocytosis of E-cadherin (Akhtar, 2001).
The ability of a cell to polarize and move is governed by remodeling of the cellular adhesion/cytoskeletal network that is in turn controlled by the Rho family of small GTPases. In fibroblasts, activation of Rho GTPases, RhoA, Rac1, and Cdc42 by different transmembrane receptors leads to distinct rearrangements of the actin cytoskeleton. Activation of RhoA stimulates actomyosin-based contractility, which leads to the assembly of actin stress fibers and focal adhesions found at the end of stress fibers. Rac1 activation leads to localized actin polymerization at the cell periphery, resulting in the formation of lamellipodia, while activation of Cdc42 results in the formation of fine
actin-rich protrusions, known as filopodia. Rac1 and Cdc42 stimulate the assembly of focal complexes that are associated with lamellipodia and filopodia, respectively. They contain a number of the same proteins found in Rho-induced focal adhesions, including vinculin, paxillin, and focal adhesion kinase. It is not known what signals lie downstream of Rac1 and Cdc42 during peripheral actin and adhesion remodeling that is required for directional migration. Individual members of the Rho family, RhoA, Rac1, and Cdc42, direct the specific intracellular targeting of c-Src tyrosine kinase to focal adhesions, lamellipodia, or filopodia, respectively. The adaptor function of c-Src (the combined SH3/SH2 domains coupled to green fluorescent protein) is sufficient for targeting. Furthermore, Src's catalytic activity is absolutely required at these peripheral cell-matrix attachment sites for remodeling that converts RhoA-dependent focal adhesions into smaller focal complexes along Rac1-induced lamellipodia (or Cdc42-induced filopodia). Consequently, cells in which kinase-deficient c-Src occupies peripheral adhesion sites exhibit impaired polarization toward migratory stimuli and reduced motility. Furthermore, phosphorylation of FAK, an Src adhesion substrate, is suppressed under these conditions. These findings demonstrate that individual Rho GTPases specify Src's exact peripheral localization and that Rac1- and Cdc42-induced adhesion remodeling and directed cell migration require Src activity at peripheral adhesion sites (Timpson, 2001).
Cadherin-dependent epithelial cell-cell adhesion is thought to be regulated by Rho family small GTPases and PI 3-kinase, but the mechanisms involved are poorly understood. Using time-lapse microscopy and quantitative image analysis, it has been shown that cell-cell contact in MDCK epithelial cells coincides with a spatio-temporal reorganization of plasma membrane Rac1 and lamellipodia from noncontacting to contacting surfaces. Within contacts, Rac1 and lamellipodia transiently concentrate at newest sites, but decrease at older, stabilized sites. Significantly, Rac1 mutants alter kinetics of cell-cell adhesion and strengthening, but not the eventual generation of cell-cell contacts. Products of PI 3-kinase activity also accumulate dynamically at contacts, but are not essential for either initiation or development of cell-cell adhesion. These results define a role for Rac1 in regulating the rates of initiation and strengthening of cell-cell adhesion (Ehrlich, 2002).
In MDCK cells, cell-cell contact is an opportunistic event that occurs when migratory cells collide. However, when contacted, cells respond rapidly and lamellipodia appear to be the primary physical drivers of cell-cell contact development. Lamellipodia become focused to the cell-cell contact zone and a region immediately surrounding it. This change coincides with an increase in Rac1 accumulation at cell-cell contacts (as determined by RacGFP localization) and a concomitant loss of Rac1 from noncontacting sites. Subsequently, RacGFP localization and lamellipodia activity becomes further restricted to newest sites in cell-cell contacts, whereas older sites have less RacGFP and membrane activity. Analysis of individual cell-cell contacts at high spatial and temporal resolution shows that Rac1 does not simply colocalize with E-cadherin/catenin complexes at cell-cell junctions as had been suggested previously, but is specifically restricted to initiating areas of contact, whereas E-cadherin gradually accumulates along the entire contact length (Ehrlich, 2002).
A detailed analysis of Rho GTPase function during vertebrate development has been undertaken by analyzing how RhoA and Rac1 control convergent extension of axial mesoderm during Xenopus gastrulation. Monitoring of a number of parameters in time-lapse recordings of mesoderm explants revealed that Rac and Rho have both distinct and overlapping roles in regulating the motility of axial mesoderm cells. The cell behaviors revealed by activated or inhibitory versions of these GTPases in native tissue are clearly distinct from those previously documented in cultured fibroblasts. The dynamic properties and polarity of protrusive activity, along with lamellipodia formation, are controlled by the two GTPases operating in a partially redundant manner, while Rho and Rac contribute separately to cell shape and filopodia formation. It is proposed that Rho and Rac operate in distinct signaling pathways that are integrated to control cell motility during convergent extension (Tahinci, 2003).
The coordinated migration of neurons is a pivotal step for functional architectural formation of the mammalian brain. To elucidate its molecular mechanism, gene transfer by means of in utero electroporation was applied in the developing murine brain, revealing the crucial roles of Rac1, its activators, STEF/Tiam1, and its downstream molecule, c-Jun N-terminal kinase (JNK), in the cerebral cortex. Functional repression of these molecules results in inhibition of radial migration of neurons without affecting their proper differentiation. Interestingly, distinct morphological phenotypes were observed; suppression of Rac1 activity causes loss of the leading process, whereas repression of JNK activity does not, suggesting the complexity of the signaling cascade. In cultured neurons from the intermediate zone, activated JNK was detected along microtubules in the processes. Application of a JNK inhibitor caused irregular morphology and increased stable microtubules in processes, and decreased phosphorylation of microtubule associated protein 1B, raising a possibility of the involvement of JNK in controlling tubulin dynamics in migrating neurons. These data thus provide important clues for understanding the intracellullar signaling machinery for cortical neuronal migration (Kawauchi, 2003).
The large extracellular polysaccharide Hyaluronan (HA) and its synthesizing enzymes (Has) have been implicated in regulating the migratory potential of metastatic cancer cells. The roles of zebrafish Has2 in normal development have been analyzed. Antisense morpholino oligonucleotide (MO)-mediated knockdown of zebrafish Has2 leads to the loss of HA, and severe migratory defects during gastrulation, somite morphogenesis and primordial germ cell migration. During gastrulation, ventrolateral cells of has2 morphant embryos fail to develop lamellipodia and to migrate dorsally, resulting in a blockage of dorsal convergence, whereas extension of the dorsal axis is normal. The effect is cell autonomous, suggesting that HA acts as an autocrine signal to stimulate the migration of HA-generating cells. Upon ectopic expression in axial cells, has2 causes the formation of supernumerary lamellipodia and a blockage of axis extension. Epistasis analyses with constitutively active and dominant-negative versions of the small GTPase Rac1 suggest that HA acts by Rac1 activation, rather than as an essential structural component of the extracellular matrix. Together, these data provide evidence that convergence and extension are separate morphogenetic movements of gastrulation. In addition, they suggest that the same HA pathways are active to auto-stimulate cell migration during tumor invasion and vertebrate embryogenesis (Bakkers, 2003).
Mesenchymal-epithelial transitions (MET) are crucial for vertebrate organogenesis. The roles of Rho family GTPases in such processes during actual development remain largely unknown. By electroporating genes into chick presomitic mesenchymal cells, it was demonstrated that Cdc42 and Rac1 play important and different roles in the MET that generates the vertebrate somites. Presomitic mesenchymal cells, which normally contribute to both the epithelial and mesenchymal populations of the somite, are hyperepithelialized when Cdc42 signaling is blocked. Conversely, cells taking up genes that elevate Cdc42 levels remain mesenchymal. Thus, Cdc42 activity levels appear critical for the binary decision that defines the epithelial and mesenchymal somitic compartments. Proper levels of Rac1 are necessary for somitic epithelialization, since cells with activated or inhibited Rac1 fail to undergo correct epithelialization. Furthermore, Rac1 appears to be required for Paraxis to act as an epithelialization-promoting transcription factor during somitogenesis (Nakaya, 2004).
A complementary pattern of phenotypes was obtained by different levels of Cdc42 activity: enhanced epithelialization and mesenchymal maintenance by inhibition and activation of Cdc42, respectively. Thus, during normal somitogenesis, different levels of Cdc42 activity appear to be critical for the binary determination during MET: Cdc42 activity needs to be low for cell epithelialization, whereas cells require high activity to maintain their mesenchymal state. Cdc42 has been reported, mainly by experiments in vitro, to assemble with several associated molecules, such as Par6, aPKC (atypical protein kinase C), and Par3 in polarizing cells. The experimental system developed in this study can be used to clarify the roles of these members in establishing epithelial structures during vertebrate morphogenesis. Recently, another member of Cdc42 subfamily, TC10, was reported to bind to N-WASP, although it remains unclear whether the binding is only to the CRIB domain. Which member among Cdc42 subfamily plays a role during somitogenesis awaits further analysis (Nakaya, 2004).
Another important finding in this study highlights differential roles between Cdc42 and Rac1 in the somitic MET. Unlike the case for Cdc42, however, overactivation and inhibition of Rac1 did not show a complementary phenotype between each other; in both cases the electroporated cells were primarily localized in the mesenchymal area, with some cells remaining in the epithelial territory. It is likely that the Rac1 activity needs to be maintained at an appropriate level to accomplish the correct MET during somitogenesis or, alternatively, that switching between the negative forms and active forms of Rac1 is important. The importance of proper Rac1 activity levels was corroborated by several lines of evidence: Rac1-activated cells that remained in the epithelial territory are not 'normal epithelial cells,' since they display aberrant accumulation of N-cadherin without polarized distribution of ZO-1. Similarly, Rac1-activated cells and Rac1-inactivated cells residing in the mesenchymal compartment also exhibit aberrantly upregulated N-cadherin and poorly organized actin polymerization, respectively. Thus, cells with an inappropriate level of Rac1 activity are neither 'normal mesenchyme' nor 'normal epithelium,' regardless of the position they occupy within a forming somite (Nakaya, 2004).
Whereas many molecules that promote cell and axonal growth cone migrations have been identified, few are known to inhibit these processes. In genetic screens designed to identify molecules that negatively regulate such migrations, CRML-1,the C. elegans homolog of CARMIL, also known as Lrrc16a, an actin-uncapping protein, was identified. Although mammalian CARMIL acts to promote the migration of glioblastoma cells, this study found that CRML-1 acts as a negative regulator of neuronal cell and axon growth cone migrations. Genetic evidence indicates that CRML-1 regulates these migrations by inhibiting the Rac GEF activity of UNC-73, a homolog of the Rac and Rho GEF Trio. The antagonistic effects of CRML-1 and UNC-73 can control the direction of growth cone migration by regulating the levels of the SAX-3 (a Robo homolog) guidance receptor. Consistent with the hypothesis that CRML-1 negatively regulates UNC-73 activity, these two proteins form a complex in vivo. Based on these observations, a role is proposed for CRML-1 as a novel regulator of cell and axon migrations that acts through inhibition of Rac signaling (Vanderzalm, 2009).
The establishment of the mammalian body plan depends on signal-regulated cell migration and adhesion, processes that are controlled by the Rho family of GTPases. This study used a conditional allele of Rac1, the only Rac gene expressed early in development, to define its roles in the gastrulating mouse embryo. Embryos that lack Rac1 in the epiblast (Rac1δepi) initiate development normally: the signaling pathways required for gastrulation are active, definitive endoderm and all classes of mesoderm are specified, and the neural plate is formed. After the initiation of gastrulation, Rac1δepi embryos have an enlarged primitive streak, make only a small amount of paraxial mesoderm, and the lateral anlage of the heart do not fuse at the midline. Because these phenotypes are also seen in Nap1 mutants, it is concluded that Rac1 acts upstream of the Nap1/WAVE complex to promote migration of the nascent mesoderm. In addition to migration phenotypes, Rac1δepi cells fail to adhere to matrix, which leads to extensive cell death. Cell death is largely rescued in Rac1δepi mutants that are heterozygous for a null mutation in Pten, providing evidence that Rac1 is required to link signals from the basement membrane to activation of the PI3K-Akt pathway in vivo. Surprisingly, the frequency of apoptosis is greater in the anterior half of the embryo, suggesting that cell survival can be promoted either by matrix adhesion or by signals from the posterior primitive streak. Rac1 also has essential roles in morphogenesis of the posterior notochordal plate (the node) and the midline (Migeotte, 2011).
Rac and cell cycle progression When microinjected into quiescent
fibroblasts, Rho, Rac, and Cdc42 stimulate cell cycle progression through G1 and subsequent DNA
synthesis. Microinjection of either the dominant negative forms of Rac and Cdc42 or of the Rho
inhibitor C3 transferase block serum-induced DNA synthesis. Unlike Ras, none of the Rho GTPases
activated the mitogen-activate protein kinase (MAPK) cascade that contains the protein kinases
c-Raf1, MEK (MAPK or ERK kinase), and ERK (extracellular signal-regulated kinase). Instead, Rac
and Cdc42, but not Rho, stimulate a distinct MAP kinase, the c-Jun kinase JNK/SAPK (Jun
NH2-terminal kinase or stress-activated protein kinase). Rho, Rac, and Cdc42 control signal
transduction pathways that are essential for cell growth (Olson, 1995).
Mammalian meiotic divisions are asymmetrical and generate a large oocyte and two small polar bodies. This asymmetry results from the anchoring of the meiotic spindle to the oocyte cortex and subsequent cortical reorganization, but the mechanisms involved are poorly understood. The role of Rac in oocyte meiosis was investigated by using a fluorescent reporter for Rac-GTP. It was found that Rac-GTP is polarized in the cortex overlying the meiotic spindle. Polarization of Rac activation occurs during spindle migration and is promoted by the proximity of chromatin to the cortex. Inhibition of Rac during oocyte maturation causes a permanent block at prometaphase I and spindle elongation. In metaphase II-arrested oocytes, Rac inhibition caused the spindle to detach from the cortex and prevented polar body emission after activation. These results demonstrate that Rac-GTP plays a major role in oocyte meiosis, via the regulation of spindle stability and anchoring to the cortex (Halet, 2007).
These data show that cytokinesis and polar body formation are under the control of Rac in mouse oocytes. A universal regulator of cytokinesis is the small GTPase RhoA, which controls the formation of the actomyosin contractile ring. In fertilized mouse oocytes, inhibition of the Rho pathway results in the formation of binucleate zygotes lacking PB2. Inhibition of Rac recapitulates this phenotype, suggesting that Rac may function upstream of Rho in mouse MII oocytes. Presumably, the polarized accumulation of Rac-GTP during meiosis I also regulates Rho-dependent cytokinesis during emission of PB1, but this could not be verified since Rac inhibition resulted in a cell cycle arrest at prometaphase (Halet, 2007).
In addition, the data suggest that Rac is required in the process of polar body protrusion. Since Rac is a well-known regulator of actin polymerization and reorganization during lamellipodium formation, it is tempting to assume that Rac plays a similar role during polar body emission. Since formation of the actin cap is unaffected by Rac inhibition, the role of Rac may rather be in reorganizing pre-existing actin filaments to induce polar body protrusion. Rac has been shown to induce myosin II phosphorylation and redistribution in neuronal cell lines, resulting in lamellipodial protrusions and cell spreading. Rac could play a similar role in oocytes by locally releasing actin filaments from their interaction with myosin II, thus decreasing cortical tension and promoting actin-mediated polar body protrusion (Halet, 2007).
An absolute requirement for asymmetrical cell division during meiosis is the maintenance of the MII spindle anchored in the oocyte cortex. These data show that this anchoring requires Rac-GTP. In oocytes lacking Rac activity, the MII spindle was found oriented perpendicular to the cortex, or completely detached from the cortex, suggesting a loss of spindle anchoring at one, and eventually at both, spindle pole. In mammalian and Xenopus oocytes, actin filaments are thought to mediate spindle anchoring, and there is evidence that actin also regulates spindle rotation before extrusion of PB2 in activated mouse oocytes. Therefore, Rac-GTP may regulate the polymerization and/or reorganization of a susbset of actin filaments necessary for spindle anchoring during the MII arrest (Halet, 2007).
In conclusion, this study identifies Rac as a major regulator of oocyte polarization and meiotic divisions. A model is proposed in which meiotic chromosomes, possibly carrying a Rac-GEF, trigger a localized activation of Rac in the oocyte cortex as a result of spindle migration. This Rac-GTP cap, in return, regulates chromosome alignment and segregation as well as polar body emission. In addition, polarized Rac-GTP serves to anchor the spindle in the cortex during the MII arrest. An important challenge for future studies is to identify the chromatin-associated factor driving Rac activation as well as the Rac effectors involved in this broad spectrum of functions (Halet, 2007).
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