EVOLUTIONARY HOMOLOGS part 1/3 || part 2/3 || part 3/3

Mushroom bodies tiny, a second PAK in Drosophila

The embryonic neurogenic phase of Drosophila brains has been studied in detail at the genetic, cellular and molecular level. In contrast, much of what is known of postembryonic brain development has been gathered by neuroanatomical and gene expression studies. The molecular mechanisms underlying cellular diversity and structural organization in the adult brain, such as the establishment of the correct neuroblast number, the spatial and temporal control of neuroblast proliferation, cell fate determination, and the generation of the precise pattern of neuronal connectivity, are largely unknown. In a screen for viable mutations affecting adult central brain structures, the mushroom bodies tiny (mbt) gene of Drosophila, which encodes a protein related to p21-activated kinase (PAK), has been isolated. Mutations in mbt primarily interfere with the generation or survival of the intrinsic cells (Kenyon cells) of the mushroom body, a paired neuropil structure in the adult brain involved in learning and memory (Melzig, 1998).

Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles

Myosin II plays a central role in epithelial morphogenesis; however, its role has mainly been examined in processes involving a single cell type. This study analyzed the structure, spatial requirement and regulation of myosin II during C. elegans embryonic elongation, a process that involves distinct epidermal cells and muscles. Novel GFP probes were developed to visualize the dynamics of actomyosin remodeling; it was found that the assembly of myosin II filaments, but not actin microfilaments, depends on the myosin regulatory light chain (MLC-4) and essential light chain (MLC-5). To determine how myosin II regulates embryonic elongation, mlc-4 mutants were rescued with various constructs and found that MLC-4 is essential in a subset of epidermal cells. Phosphorylation of two evolutionary conserved MLC-4 serine and threonine residues is important for myosin II activity and organization. In an RNAi screen for potential myosin regulatory light chain kinases, it was found that the ROCK, PAK and MRCK homologs act redundantly. The combined loss of ROCK and PAK, or ROCK and MRCK, completely prevented embryonic elongation, but a constitutively active form of MLC-4 could only rescue a lack of MRCK. This result, together with systematic genetic epistasis tests with a myosin phosphatase mutation, suggests that ROCK and MRCK regulate MLC-4 and the myosin phosphatase. Moreover, it is suggested that ROCK and PAK regulate at least one other target essential for elongation, in addition to MLC-4 (Gally, 2009).

Identification of novel mammalian PAKs

The androgen receptor (AR) is a hormone-dependent transcription factor that plays important roles in male sexual differentiation and development. Transcription activation by steroid hormone receptors, such as the androgen receptor, is mediated through interaction with cofactors. A novel AR-interacting protein, provisionally termed PAK6, has been identified that shares a high degree of sequence similarity with p21-activated kinases (PAKs). PAK6 is a 75-kDa protein that contains a putative amino-terminal Cdc42/Rac interactive binding motif and a carboxyl-terminal kinase domain. A domain-specific and ligand-dependent interaction between AR and PAK6 was further confirmed in vivo and in vitro. Northern blot analysis has revealed that PAK6 is highly expressed in testis and prostate tissues. Most importantly, immunofluorescence studies show that PAK6 cotranslocates into the nucleus with AR in response to androgen. Transient transfection experiments show that PAK6 specifically represses AR-mediated transcription. This report identifies a novel function for a PAK-homologous protein and suggests a potential unique mechanism by which other signal transduction pathways may cross-talk with AR pathways to regulate AR function in normal and malignant prostate cells (Yang, 2001).

A novel human PAK family kinase, PAK5, has been cloned. PAK5 contains a CDC42/Rac1 interactive binding (CRIB) motif at the N-terminus and a Ste20-like kinase domain at the C-terminus. PAK5 is structurally most related to PAK4 and PAK6 to make up the PAK-II subfamily. PAK5 preferentially binds to CDC42 in the presence of GTP and the CRIB motif is essential for this interaction. PAK5 is a functional protein kinase but unlike PAK-I family kinases (PAK1, 2, and 3), the kinase activity of PAK5 does not seem to require the binding of CDC42. Overexpression of PAK5 activates the JNK kinase pathway but not p38 or ERK pathways. PAK5 transcript is predominantly expressed in brain as revealed by Northern blot and in situ hybridization. The expression pattern of PAK5 is distinct from that of PAK4 and PAK6, suggesting a functional division among PAK-II subfamily kinases based on differential tissue distribution (Pandey, 2002).

Structural studies of PAKs

Rho (see Drosophila Rho1), Rac (see Drosophila Rac1), and Cdc42 are small GTPases that regulate the formation of a variety of actin structures and the assembly of associated integrin complexes, but little is known about the target proteins that mediate their effects. A motif-based search method has been used to identify putative effector proteins for Rac and Cdc42. A search of the GenBankTM data base for similarity with the minimum Cdc42/Rac interactive binding (CRIB) region of a potential effector protein p65PAK has identified over 25 proteins containing a similar motif from a range of different species. These candidate Cdc42/Rac-binding proteins include family members of the mixed lineage kinases (MLK); a novel tyrosine kinase from Drosophila melanogaster (DPR2, Fak-like tyrosine kinase); a human protein MSE55, and several novel yeast and Caenorhabditis elegans proteins. Two murine p65PAK isoforms and a candidate protein from C. elegans, F09F7.5, interact strongly with the GTP form of both Cdc42 and Rac, but not Rho in a filter binding assay. Three additional candidate proteins, DPR2, MSE55, and MLK3 show binding to the GTP form of Cdc42 and weaker binding with Rac, and again no interaction with Rho. These results indicate that proteins containing the CRIB motif bind to Cdc42 and/or Rac in a GTP-dependent manner, and they may, therefore, participate in downstream signaling (Burbelo, 1995).

betaPix (PAK-interacting exchange factor) is a recently identified guanine nucleotide exchange factor for Rho family small G protein Cdc42/Rac. The protein interacts with p21-activated protein kinase (PAK) through its SH3 domain. The effect of betaPix on MAP kinase signaling and cytoskeletal rearrangement has been studied in NIH3T3 fibroblast cells. Overexpression of betaPix enhances the activation of p38 in the absence of other stimuli and also induces translocation of p38 to the nucleus. This betaPix-induced p38 activation is blocked by coexpression of dominant-negative Cdc42/Rac or kinase-inactive PAK, indicating that the effect of betaPix on p38 is exerted through the Cdc42/Rac-PAK pathway and requires PAK kinase activity. The essential role of betaPix in growth factor-stimulated p38 activation is evidenced by the blocking of platelet-derived growth factor-induced p38 activation in the cells expressing betaPix SH3m (W43K) and betaPix DHm (L238R,L239R). In addition, SB203580, a p38 inhibitor, and kinase-inactive p38 (T180A,Y182F) block membrane ruffling induced by betaPix, suggesting that p38 might be involved in mediating betaPix-induced membrane ruffling. The results in this study suggest that betaPix might have a role in nuclear signaling, as well as in actin cytoskeleton regulation, and that some part of these cellular functions is possibly mediated by p38 MAP kinase (Lee, 2001).

p21-activated kinases (PAKs) are implicated in integrin signalings, and have been proposed to associate with paxillin indirectly. Paxillin can bind directly to PAK3. Several representative focal adhesion proteins were examined, and it was found that paxillin is the sole protein that associates with PAK3. PAK3 associates with the alpha and beta isoforms of paxillin, but not with gamma. Paxillin alpha associates with both the kinase-inactive and the Cdc42-activated forms of PAK3 in vivo, without affecting the activation states of the kinase. A number of different functions have been ascribed to PAKs; and PAKs can bind directly to growth factor signaling-adaptor molecule, Nck, and a guanine nucleotide exchanger, betaPIX. Paxillin alpha can compete with Nck and betaPIX in the binding of PAK3. Moreover, paxillin alpha can be phosphorylated by PAK3 at serine. Therefore, paxillin alpha, but not gamma, appears to be capable of linking both the kinase-inactive and activated forms of PAK3 to integrins independent of Nck and betaPIX, because Nck links PAK1 to growth factor receptors. These results also reveal that paxillin is involved in highly complexed protein-protein interactions in integrin signaling (Hashimoto, 2001).

Guanine nucleotide exchange factors in the Dbl family activate Rho GTPases by accelerating dissociation of bound GDP, promoting acquisition of the GTP-bound state. Dbl proteins possess a approximately 200 residue catalytic Dbl-homology (DH) domain that is arranged in tandem with a C-terminal pleckstrin homology (PH) domain in nearly all cases. The solution structure of the DH domain of human PAK-interacting exchange protein (betaPIX: see Drosophila rho-type guanine exchange factor) is reported here. The domain is composed of 11 alpha-helices that form a flattened, elongated bundle. The structure explains a large body of mutagenesis data, which, along with sequence comparisons, identify the GTPase interaction site as a surface formed by three conserved helices near the center of one face of the domain. Proximity of the site to the DH C-terminus suggests a means by which PH-ligand interactions may be coupled to DH-GTPase interactions to regulate signaling through the Dbl proteins in vivo (Aghazadeh, 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 that result 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), though not affecting Cdc42 binding, produced a constitutively active kinase, suggesting 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 the p21. Coexpression of PAK inhibitor with Rac1G12V also prevents loss of stress fibers but not ruffling induced by the 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 observations with constitutively active PAK, demonstrate that these kinases play an important role downstream of Cdc42 and Rac1 in cytoskeletal reorganization (Zhao, 1998).

The p21-activated kinases (PAKs), stimulated by binding with GTP-liganded forms of Cdc42 or Rac, modulate cytoskeletal actin assembly and activate MAP-kinase pathways. The 2.3 Å resolution crystal structure of a complex between the N-terminal autoregulatory fragment and the C-terminal kinase domain of PAK1 shows that GTPase binding will trigger a series of conformational changes, beginning with disruption of a PAK1 dimer and ending with rearrangement of the kinase active site into a catalytically competent state. An inhibitory switch (IS) domain, which overlaps the GTPase binding region of PAK1, positions a polypeptide segment across the kinase cleft. GTPase binding will refold part of the IS domain and unfold the rest. A related switch has been seen in WASP, the Wiskott-Aldrich syndrome protein (Lei, 2000).

p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491) that is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity [K(d) 0.6 microM] to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher [K(d) 18 nM], suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation (Buchwald, 2001).

Pak1, a serine/threonine kinase that regulates the actin cytoskeleton, is an effector of the Rho family GTPases Cdc42 and Rac1. The crystal structure of Pak1 revealed an autoinhibited dimer that must dissociate upon GTPase binding. Pak1 forms homodimers in vivo and its dimerization is regulated by the intracellular level of GTP-Cdc42 or GTP-Rac1. The dimerized Pak1 adopts a trans-inhibited conformation: the N-terminal inhibitory portion of one Pak1 molecule in the dimer binds and thereby inhibits the catalytic domain of the other molecule. One GTPase interaction can result in activation of both partners. Another ligand, ßPIX, can stably associate with dimerized Pak1. Dimerization does not facilitate Pak1 trans-phosphorylation. It is concluded that the functional significance of dimerization is to allow trans-inhibition (Parrini, 2002).

The precise temporal-spatial regulation of the p21-activated serine-threonine kinase PAK at the plasma membrane is required for proper cytoskeletal reorganization and cell motility. However, the mechanism by which PAK localizes to focal adhesions has not yet been elucidated. Indirect binding of PAK to the focal adhesion protein paxillin via the Arf-GAP protein paxillin kinase linker (PKL) and PIX/Cool suggested a mechanism. This study demonstrates an essential role for a paxillin-PKL interaction in the recruitment of activated PAK to focal adhesions. Similar to PAK, expression of activated Cdc42 and Rac1, but not RhoA, stimulates the translocation of PKL from a generally diffuse localization to focal adhesions. Expression of the PAK regulatory domain (PAK1-329) or the autoinhibitory domain (AID 83-149) induces PKL, PIX, and PAK localization to focal adhesions, indicating a role for PAK scaffold activation. PIX, but not NCK, binding to PAK is necessary for efficient focal adhesion localization of PAK and PKL, consistent with a PAK-PIX-PKL linkage. Although PAK activation is required, it is not sufficient for localization. The PKL amino terminus, containing the PIX-binding site, but lacking paxillin-binding subdomain 2 (PBS2), is unable to localize to focal adhesions and also abrogates PAK localization. An identical result was obtained after PKLDeltaPBS2 expression. Finally, neither PAK nor PKL is capable of localizing to focal adhesions in cells overexpressing paxillinDeltaLD4, confirming a requirement for this motif in recruitment of the PAK-PIX-PKL complex to focal adhesions. These results suggest a GTP-Cdc42/GTP-Rac triggered multistep activation cascade leading to the stimulation of the adaptor function of PAK, which through interaction with PIX provokes a functional PKL PBS2-paxillin LD4 association and consequent recruitment to focal adhesions. This mechanism is probably critical for the correct subcellular positioning of PAK, thereby influencing the ability of PAK to coordinate cytoskeletal reorganization associated with changes in cell shape and motility (Brown, 2002).

Activation and inactivation of PAKs

Studies in Dictyostelium have shown that the p110-related phosphatidylinositol-3-kinases PI3K1 and PI3K2 are required for proper development, pinocytosis chemotaxis, and chemoattractant-mediated activation of PKB. Insights into the mechanism by which PI3K regulates chemotaxis derive from studies on PKB in mammalian leukocytes and Dictyostelium cells. PKB activation requires its translocation to the plasma membrane by binding of its PH domain to PtdIns(3,4,5)P3 and PtdIns(3,4)P2 produced upon activation of PI3K, leading to PKB activation. In leukocytes and Dictyostelium cells, chemoattractants mediate PKB activation through a G-protein-coupled pathway that requires the activity of the respective PI3Ks. Chemoattractant stimulation of neutrophils and Dictyostelium cells results in a transient localization of a GFP fusion of the PH domains from the Dictyostelium and mammalian PKBs to the plasma membrane. When these cells are placed in a chemoattractant gradient, membrane localization of the PKB-PH-GFP fusion is restricted to the leading edge, as is the case for other PH-domain-containing proteins in Dictyostelium. In Dictyostelium, translocation of the PKB-PH domain GFP fusion is PI3K-dependent. PI3 kinase and protein kinase B (PKB or Akt) control cell polarity and chemotaxis, in part, through the regulation of PAKa, a structural homolog of mammalian PAKs (p21-activated kinase) that is required for myosin II assembly. PI3K and PKB mediate PAKa's subcellular localization, PAKa's activation in response to chemoattractant stimulation, and chemoattractant-mediated myosin II assembly. Mutation of the PKB phosphorylation site in PAKa to Ala blocks PAKa's activation and inhibits PAKa redistribution in response to chemoattractant stimulation, whereas an Asp substitution leads to an activated protein. Addition of the PI3K inhibitor LY294002 results in a rapid loss of cell polarity and the axial distribution of actin, myosin, and PAKa. These results provide a mechanism by which PI3K regulates chemotaxis (Chung, 2001).

Serine/threonine protein kinases of the Ste20/PAK family have been implicated in the signaling 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 for a role of these kinases in Gbetagamma-mediated signal transduction in organisms ranging from yeast to mammals (Leeuw, 1998).

Nck (Drosophila homolog: Dreadlocks) is an adaptor protein composed of a single SH2 domain and three SH3 domains. Upon growth factor stimulation, Nck is recruited to receptor tyrosine kinases via its SH2 domain, probably initiating one or more signaling cascades. Nck is bound in living cells to the serine-threonine kinase Pak1. The association between Nck and Pak1 is mediated by the second SH3 domain of Nck and a proline-rich sequence in the amino terminus of Pak1. Pak1 is recruited by activated epidermal growth factor (EGF) and platelet-derived growth factor receptors. Moreover, Pak1 kinase activity is increased in response to EGF in HeLa cells transfected with human Pak1, and the kinase activity is enhanced when Nck is co-transfected. It is concluded that Nck links receptor tyrosine kinases with Pak1 and is probably involved in targeting and regulation of Pak1 activity (Galisteo, 1996).

The adaptor protein Nck consists of three Src homology 3 (SH3) domains followed by one SH2 domain. Like the Grb2 adaptor protein, which is known to couple receptor tyrosine kinases to the small GTPase Ras, Nck is presumed to bind to tyrosine-phosphorylated proteins using its SH2 domain and to downstream effector proteins using its SH3 domain. Little is known, however, about the specific biological function of Nck. The Pak family of serine/threonine kinases are known to be activated by binding to the GTP-bound form of Cdc42 or Rac1, which are small GTPases of the Rho family that are involved in regulating the organization of the actin cytoskeleton. Evidence is presented that Nck can mediate the relocalization and subsequent activation of the Pak1 kinases. Nck associates in vivo with Pak, using the second of Nck's three SH3 domains; localization of this individual Nck SH3 domain, or of Pak kinase itself, to the membrane results in activation of Pak and stimulation of downstream mitogen activated protein kinase cascades. Activation of downstream signaling by the membrane-localized Nck SH3 domain is blocked by a kinase-inactive mutant form of Pak1. These results demonstrate that localization of Pak1 to the membrane in the absence of other signals is sufficient for its activation, and imply that the Nck adaptor protein could function to link changes in tyrosine phosphorylation of cellular proteins to the Cdc42/Pak signaling pathway (Lu, 1997).

Pak1 can be targeted to the membrane by Nck in response to tyrosine phosphorylation, and membrane association of Pak1 is sufficient to increase its specific activity. The mechanism whereby Pak is activated by membrane localization, however, is unknown. Expression of three proteins that inhibit Rho-family GTPases by different mechanisms (RhoGDI, Bcr and D57Y Cdc42) all block the activation of Pak by a membrane-targeted Nck SH3 domain, demonstrating that the in vivo activation of Pak1 induced by membrane localization is dependent on Rho-family GTPases. This implies that Pak activity can be regulated in cells both by the level of GTP loading of various Rho-family GTPases and the local concentration of Pak relative to these GTPases. These data also suggest the existence of Rho-family GTPases in addition to Cdc42 and Rac1 that can activate Pak on membranes (Lu, 1999).

Activation of p21-activated kinases (Paks) is achieved through binding of the GTPases Rac or Cdc42 to a conserved domain in the N-terminal regulatory region of Pak. Additional signaling components are also likely to be important in regulating Pak activation. Recently, a family of Pak-interacting guanine nucleotide exchange factors (Pix) has been identified whose members are good candidates for regulating Pak activity. Using an active, truncated form of alphaPix (amino acids 155-545), stimulation of Pak1 kinase activity is observed when alphaPix155-545 is co-expressed with Cdc42 and wild-type Pak1 in COS-1 cells. This activation does not occur when a Pak1 mutant unable to bind alphaPix is coexpressed. The activation of wild-type Pak1 by alphaPix155-545 also requires that alphaPix155-545 retain functional exchange factor activity. However, the Pak1(H83,86L) mutant that does not bind Rac or Cdc42 is activated in the absence of GTPase by alphaPix155-545 and by a mutant of alphaPix155-545 that no longer has exchange factor activity. Pak1 activity stimulated in vitro using GTPgammaS-loaded Cdc42 is also enhanced by recombinant alphaPix155-545 in a binding-dependent manner. These data suggest that Pak activity can be modulated by physical interaction with alphaPix and that this specific effect involves both exchange factor-dependent and -independent mechanisms (Daniels, 1999).

Pak1 protein kinase of Schizosaccharomyces pombe, a member of the p21-GTPase-activated protein kinase (PAK) family, participates in signaling pathways including sexual differentiation and morphogenesis. The regulatory domain of PAK proteins is thought to inhibit the kinase catalytic domain, as truncation of this region renders kinases more active. The interaction between Pak1 regulatory domain and the kinase catalytic domain has been detected in the two-hybird system. Pak1 catalytic domain binds to the same highly conserved region on the regulatory domain that binds Cdc42, a GTPase protein capable of activating Pak1. Two-hybrid, mutant, and genetic analyses indicate that this intramolecular interaction renders the kinase in a closed and inactive configuration. Cdc42 can induce an open configuration of Pak1. It is proposed that Cdc42 interaction disrupts the intramolecular interactions of Pak1, thereby releasing the kinase from autoinhibition (Tu, 1999).

A potent negative regulator of Ste20p is described in the Saccharomyces cerevisiae filamentous growth-signaling pathway. A mutant, hsl7, was examined that exhibits filamentous growth on rich medium. Hsl7p belongs to a highly conserved protein family in eukaryotes. Hsl7p associates with the noncatalytic region within the amino-terminal half of Ste20p as well as Cdc42p. Deletions of HSL7 in haploid and diploid strains lead to cell elongation and enhancement of both haploid invasive growth and diploid pseudohyphal growth. However, deletions of STE20 in haploid and diploid cells greatly diminishes these hsl7-associated phenotypes. In addition, overexpression of HSL7 inhibits pseudohyphal growth. Thus, Hsl7p may inhibit the activity of Ste20p in the S. cerevisiae filamentous growth-signaling pathway. This genetic analyses suggests the possibility that Cdc42p and Hsl7p compete for binding to Ste20p for pseudohyphal development when starved for nitrogen (Fujita 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 Rho GTPases are involved in many signaling pathways and cellular functions, including the organization of the actin cytoskeleton, regulation of transcription, cell motility, and cell division. The p21 (Cdc42/Rac)-activated kinase PAK mediates a number of biological effects downstream of these Rho GTPases. The phosphorylation state of mammalian PAK is highly regulated: upon binding of GTPases, PAK is potently activated by autophosphorylation at multiple sites, although the mechanisms of PAK downregulation are not known. Two PP2C-like serine/threonine phosphatases (POPX1 and POPX2) efficiently inactivate PAK. POPX1 was isolated as a binding partner for the PAK interacting guanine nucleotide exchange factor PIX. The dephosphorylating activity of POPX correlates with an ability to block the in vivo effects of active PAK. Consonant with these effects on PAK, POPX can also inhibit actin stress fiber breakdown and morphological changes driven by active Cdc42V12. The association of the POPX phosphatases with PAK complexes may allow PAK to cycle rapidly between active and inactive states; it represents a unique regulatory component of the signaling pathways of the PAK kinase family (Koh, 2002).

The small GTPase Rac has been implicated in growth cone guidance mediated by semaphorins and their receptors. Plexin-B1, a receptor for Semaphorin4D (Sema4D), and p21-activated kinase (PAK) can compete for the interaction with active Rac and plexin-B1 can inhibit Rac-induced PAK activation. Expression of active Rac enhances the ability of plexin-B1 to interact with Sema4D. Active Rac stimulates the localization of plexin-B1 to the cell surface. The enhancement in Sema4D binding depends on the ability of Rac to bind plexin-B1. These observations support a model where signaling between Rac and plexin-B1 is bidirectional; Rac modulates plexin-B1 activity and plexin-B1 modulates Rac function (Vikis, 2002).

Sema4D enhances the interaction between plexin-B1 and active Rac. A model is proposed by which Sema4D binds the plexin-B1 receptor and stimulates the recruitment of Rac-GTP. Sequestration of Rac results in the inactivation of PAK and growth cone collapse/turning. This model conflicts with studies on the role of Rac downstream of the plexin-A1 receptor where dominant negative Rac inhibits collapse in response to Sema3A; this suggests that Rac activation is required for Sema3A-mediated growth cone collapse. Perhaps plexin-A and -B signal via different mechanisms since plexin-A does not interact with active Rac. However, in Drosophila, Rac functions downstream of plexA even though it does not interact with plexA. It is possible that a yet unidentified protein couples plexin-A with Rac (Vikis, 2002).

The p21-activated kinases (PAKs), in common with many kinases, undergo multiple autophosphorylation events upon interaction with appropriate activators. The Cdc42-induced phosphorylation of PAK serves in part to dissociate the kinase from its partners PIX and Nck. How autophosphorylation events affect the catalytic activity of PAK has been investigated in detail by altering the autophosphorylation sites in both alpha- and betaPAK. Both in vivo and in vitro analyses demonstrate that, although most phosphorylation events in the PAK N-terminal regulatory domain play no direct role in activation, a phosphorylation of alphaPAK serine 144 or betaPAK serine 139, which lie in the kinase inhibitory domain, significantly contribute to activation. By contrast, sphingosine-mediated activation is independent of this residue, indicating a different mode of activation. Thus two autophosphorylation sites direct activation while three others control association with focal complexes via PIX and Nck (Chong, 2001).

The Nf2 tumor suppressor gene codes for merlin, a protein whose function has been elusive. A novel interaction is described between merlin and p21-activated kinase 1 (Pak1), which is dynamic and facilitated upon increased cellular confluence. Merlin inhibits the activation of Pak1, as evidenced by the observation that the loss of merlin expression results in the inappropriate activation of Pak1 under conditions associated with low basal activity. Conversely, the overexpression of merlin in cells that display a high basal activity of Pak1 results in the inhibition of Pak1 activation. This inhibitory function of merlin is mediated through its binding to the Pak1 PBD and by inhibiting Pak1 recruitment to focal adhesions. This link provides a possible mechanism for the effect of loss of merlin expression in tumorigenesis (Kissil, 2003).

The p21-activated kinases (PAKs) are important mediators of cytoskeletal reorganization, cell motility and transcriptional events regulated by the Rho family GTPases Rac and Cdc42. PAK activation by serum components is strongly dependent on cell adhesion to the extracellular matrix (ECM). PAK binds directly to the Nck adapter protein, an interaction thought to play an important role in regulation and localization of PAK activity. The interaction of PAK with Nck is regulated dynamically by cell adhesion. PAK-Nck binding is rapidly lost after cell detachment and rapidly restored after re-adhesion to the ECM protein fibronectin, suggesting a rapidly reversible mode of regulation. Furthermore, the loss of Nck binding correlates with changes in the phosphorylation state of PAK in nonadherent cells, as evidenced by electrophoretic mobility shift and phosphorylation within a sequence known to mediate interaction with Nck. The ability of cell adhesion to regulate PAK phosphorylation and interaction with Nck may contribute to the anchorage-dependence of PAK activation as well as to the localization of activated PAK within a cell.

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.

Ras may activate PAK

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. Furthermore, 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 are 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).

Neurofibromatosis type 1 (NF1), a common autosomal dominant disorder caused by loss of the NF1 gene, is characterized clinically by neurofibromas and more rarely by neurofibrosarcomas. Neurofibromin (see Drosophila Neurofibromin 1), the protein encoded by NF1, possesses an intrinsic GTPase accelerating activity for the Ras proto-oncogene. Through this activity, neurofibromin is a negative regulator of Ras. The Pak protein kinase is a candidate for a downstream signaling protein that may mediate Ras signals because it is activated by Rac and Cdc42, two small G proteins required for Ras signaling. Pak mutants have been used to explore the role of Pak in Ras signaling in Schwann cells, the cells affected in NF1. Whereas an activated Pak mutant does not transform cells, dominant negative Pak mutants are potent inhibitors of Ras transformation of rat Schwann cells and of a neurofibrosarcoma cell line from an NF1 patient. Although activated Pak stimulates jun-N-terminal kinase, inhibition of Ras transformation by dominant negative Pak does not require inhibition of jun-N-terminal kinase. Instead, the Pak mutants appear to inhibit transformation by preventing Ras activation of the ERK/mitogen-activated protein kinase cascade. These results have implications for understanding of NF1 because a neurofibrosarcoma cell line derived from a patient with NF1 was reverted by stable expression of the Pak dominant negative mutants (Tang, 1998).

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), which, 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 have suggested 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 do activate Pak when they are cotransfected. Similarly, RacV12H40, an effector mutant that does not bind Pak, and Rho both cooperated 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 one another 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).

Activation of the protein kinase Raf-1 is a complex process involving association with the GTP-bound form of Ras (Ras-GTP), membrane translocation and both serine/threonine and tyrosine phosphorylation. p21-activated kinase 3 (Pak3) upregulates Raf-1 through direct phosphorylation on Ser338. The origin of the signal for Pak-mediated Raf-1 activation has been investigated by examining the roles of the small GTPase Cdc42, Rac and Ras, and of phosphatidylinositol (PI) 3-kinase. Pak3 acts synergistically with either Cdc42V12 or Rac1V12 to stimulate the activities of Raf-1, Raf-CX, a membrane-localized Raf-1 mutant, and Raf-1 mutants defective in Ras binding. Raf-1 mutants defective in Ras binding are also readily activated by RasV12. This indirect activation of Raf-1 by Ras is blocked by a dominant-negative mutant of Pak, implicating an alternative Ras effector pathway in Pak-mediated Raf-1 activation. Pak-mediated Raf-1 activation is upregulated by both RasV12C40, a selective activator of PI 3-kinase, and p110-CX, a constitutively active PI 3-kinase. In addition, p85delta, a mutant of the PI 3-kinase regulatory subunit, inhibits the stimulated activity of Raf-1. Pharmacological inhibitors of PI 3-kinase also block both activation and Ser338 phosphorylation of Raf-1 induced by epidermal growth factor (EGF). Thus, Raf-1 activation by Ras is achieved through a combination of both physical interaction and indirect mechanisms involving the activation of a second Ras effector, PI 3-kinase, which directs Pak-mediated regulatory phosphorylation of Raf-1 (Sun, 2000).

Erbin and the NF2 tumor suppressor Merlin cooperatively regulate cell-type-specific activation of PAK2 by TGF-beta

Transforming growth factor beta (TGF-beta) family ligands are pleotropic proteins with diverse cell-type-specific effects on growth and differentiation. For example, PAK2 activation is critical for the proliferative/profibrotic action of TGF-beta on mesenchymal cells, and yet it is not responsive to TGF-beta in epithelial cells. Therefore this study investigated the regulatory constraints that prevent inappropriate PAK2 activation in epithelial cultures. The results show that the epithelial-enriched protein Erbin controls the function of the NF2 tumor suppressor Merlin by determining the output of Merlin's physical interactions with active PAK2. Whereas mesenchymal TGF-beta signaling induces PAK2-mediated inhibition of Merlin function in the absence of Erbin, Erbin/Merlin complexes bind and inactivate GTPase-bound PAK2 in epithelia. These results not only identify Erbin as a key determinant of epithelial resistance to TGF-beta signaling, they also show that Erbin controls Merlin tumor suppressor function by switching the functional valence of PAK2 binding (Wilkes, 2009).

DSCAM interacts with and activates Pak

DSCAM is a member of the immunoglobulin superfamily that maps to a Down syndrome region of chromosome 21q22.2-22.3. Genetic and biochemical studies have shown that in Drosophila, Dscam activates Pak1 via the Dock adaptor molecule. The extracellular domain of human DSCAM is highly homologous to the Drosophila protein; however, the intracellular domains of both human and Drosophila DSCAM share no obvious sequence identity. To study the signaling mechanisms of human DSCAM, the interaction between DSCAM and potential downstream molecules was investigated. DSCAM was shown to directly bind to Pak1 and stimulates Pak1 phosphorylation and activity, unlike Drosophila, where an adaptor protein Dock mediates the interaction between Dscam and Pak1. DSCAM activates both JNK and p38 MAP kinases. Furthermore, expression of the cytoplasmic domain of DSCAM induces a morphological change in cultured cells that is JNK-dependent. These observations suggest that human DSCAM also signals through Pak1 and may function in axon guidance similar to the Drosophila Dscam (Li, 2004).

Inca: a novel p21-activated kinase-associated protein required for cranial neural crest development

Inca (induced in neural crest by AP2) is a novel protein discovered in a microarray screen for genes that are upregulated in Xenopus embryos by the transcriptional activator protein Tfap2a. It has no significant similarity to any known protein, but is conserved among vertebrates. In Xenopus, zebrafish and mouse embryos, Inca is expressed predominantly in the premigratory and migrating neural crest (NC). Knockdown experiments in frog and fish using antisense morpholinos reveal essential functions for Inca in a subset of NC cells that form craniofacial cartilage. Cells lacking Inca migrate successfully but fail to condense into skeletal primordia. Overexpression of Inca disrupts cortical actin and prevents formation of actin 'purse strings', which are required for wound healing in Xenopus embryos. Inca physically interacts with p21-activated kinase 5 (PAK5), a known regulator of the actin cytoskeleton that is co-expressed with Inca in embryonic ectoderm, including in the NC. These results suggest that Inca and PAK5 cooperate in restructuring cytoskeletal organization and in the regulation of cell adhesion in the early embryo and in NC cells during craniofacial development (Luo, 2007).

PAK function in directional sensing

Efficient chemotaxis requires directional sensing and cell polarization. A signaling mechanism is described involving Gβγ, PAK-associated guanine nucleotide exchange factor (PIXα), Cdc42, and p21-activated kinase (PAK) 1. This pathway is utilized by chemoattractants to regulate directional sensing and directional migration of myeloid cells. The results suggest that Gβγ binds PAK1 and, via PAK-associated PIXα, activates Cdc42, which in turn activates PAK1. Thus, in this pathway, PAK1 is not only an effector for Cdc42, but it also functions as a scaffold protein required for Cdc42 activation. This Gβγ-PAK1/PIXα/Cdc42 pathway is essential for the localization of F-actin formation to the leading edge, the exclusion of PTEN from the leading edge, directional sensing, and the persistent directional migration of chemotactic leukocytes. Although ligand-induced production of PIP3 is not required for activation of this pathway, PIP3 appears to localize the activation of Cdc42 by the pathway (Li, 2003).

Chemoattractants play a central role in regulation of inflammatory reactions by attracting and activating leukocytes at sites of injury and inflammation. Most chemoattractants bind to serpentine cell surface receptors that activate the Gi family of G proteins to elicit a range of responses in leukocytes that includes chemotaxis. Chemotaxis is an intriguing biological process in which cells interpret gradients of chemoattractants to move toward higher concentrations of chemoattractants. During chemotaxis, chemoattractants elicit a number of changes in cells. These include morphological changes characterized by cell elongation and formation of lamellae at the leading edge. There are also biochemical changes characterized by the polarized distribution of some intracellular proteins. Many of these processes depend on cytoskeleton reorganization. The Rho GTPase family of small G proteins has been demonstrated to play a key role in mediating extracellular stimulus-induced cytoskeleton reorganization, often by stimulating the formation of various polymerized actin structures (Li, 2003).

Cdc42 has been shown to interact with and/or activate proteins known to be involved in cytoskeleton reorganization, including the PAK kinases and Wiscott Aldrich syndrome protein (WASP). However, the precise mechanisms by which Cdc42 regulates cytoskeleton reorganization are not clear. There are three highly homologous PAK isoforms that contain a C-terminal kinase domain, a PIX binding domain, several proline-rich domains, and an autoregulatory segment. The autoregulatory segment contains a CRIB (Cdc42/Rac interaction/binding) domain, dimerization sequences, an inhibitory switch domain, and a kinase inhibition sequence. The PIX domain binds with a very high affinity to PIX guanine nucleotide exchange factors (GEFs), consisting of PIXα and PIXβ (also called Cool1 and Cool2, respectively). PAK and PIX proteins are readily coimmunoprecipitated from unstimulated cells, suggesting that these two proteins are normally associated. PIXα has been shown to stimulate nucleotide exchange activity for Cdc42 and Rac in vivo and in vitro (Li, 2003 and references therein).

PAK1 protein forms a homodimer. The binding of GTP bound Cdc42 or Rac dissociates the PAK dimer, leading to PAK activation. In addition, translocation of PAK proteins to membranes, such as that translocation mediated by binding to Nck, moderately activates PAK kinase activity. One effect of PAK1 activation is the regulation of actin cytoskeletal organization and dynamics, which may underlie PAK's role in regulating cell migration and axon guidance (Li, 2003).

Chemoattractants have been shown to activate Rac, Cdc42, and PAK kinases in leukocytes. The recent discovery of P-Rex1 [a novel Rac activator serving as a Gβγ and phosphatidylinositol (3,4,5) triphosphate (PIP3)-dependent GEF for Rac], suggests that chemoattractants may regulate Rac via P-Rex1; however, the mechanisms by which chemoattractants regulate Cdc42 remain elusive. In this report, a signaling mechanism has been identified by which Gβγ directly interacts with PAK1 and activates Cdc42 through PAK1-associated PIXα. Activated Cdc42 in turn activates PAK1. Utilizing short interference RNA (siRNA)-mediated knockdown and mouse gene-targeting approaches, it has been demonstrated that chemoattractants use this pathway to specifically activate Cdc42, but not Rac, in myeloid cells and reveal an essential role for this signaling pathway in establishing cell directionality during chemotaxis (Li, 2003).

Cadherin adhesion, tissue tension, and noncanonical Wnt signaling regulate fibronectin matrix organization

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).

A Trio-Rac1-Pak1 signalling axis drives invadopodia disassembly

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).

PAK and synaptic morphology

Molecular and cellular mechanisms for memory consolidation in the cortex are poorly known. To study the relationships between synaptic structure and function in the cortex and consolidation of long-term memory, transgenic mice have been generated in which catalytic activity of PAK, a critical regulator of actin remodeling, is inhibited in the postnatal forebrain. Cortical neurons in these mice display fewer dendritic spines and an increased proportion of larger synapses when compared to wild-type controls. These alterations in basal synaptic morphology correlate with enhanced mean synaptic strength and impaired bidirectional synaptic modifiability (enhanced LTP and reduced LTD) in the cortex. By contrast, spine morphology and synaptic plasticity are normal in the hippocampus of these mice. Importantly, these mice exhibit specific deficits in the consolidation phase of hippocampus-dependent memory. Thus, these results provide evidence for critical relationships between synaptic morphology and bidirectional modifiability of synaptic strength in the cortex and consolidation of long-term memory (Hayashi, 2004).

Spinogenesis occurs not only during development of the brain but also in adulthood in response to plasticity-inducing stimuli. A role for PAK in developmental spinogenesis was suggested by a recent in vitro study in which transfection of a dnPAK construct into cultured 10-day-old hippocampal neurons was shown to block ephrin B-induced spine development. The lower spine density that was observed in the cortex of dnPAK transgenic mice could in part reflect PAK's role in developmental spinogenesis. However, it is believed that the contribution of developmental effects should be minimal, since endogenous PAK activity is not detectably inhibited in the transgenic mice through the end of the third postnatal week, by which time developmental spinogenesis is nearly complete in the cortex. Therefore, it is likely that the lower spine density in transgenic mice indicates a critical role of PAK in activity-induced spinogenesis in the adult brain. Consistent with this role, it was found that, in mature neurons, active PAK is associated with the PSD of spines and is elevated upon NMDAR activation (Hayashi, 2004).

Through its function in actin remodeling, PAK might mediate de novo spine formation and/or spine duplication: the two proposed modes of activity-dependent spinogenesis. During de novo spine formation, active PAK could be required for the formation of new protrusions from dendritic shafts, perhaps by promoting actin polymerization via phosphorylation of LIM kinase and inactivation of cofilin/actin-depolymerizing factor. In this case, the increased proportion of larger perforated spines (which usually represent stronger synapses) that is observed in dnPAK transgenic mice could be explained as a consequence of homeostatic compensation for reduced spine number, in order to maintain the overall output of individual neurons. Alternatively, during spine duplication in which preexisting spines enlarge, undergo PSD perforation, and split to form new simple spines, active PAK could be required for the splitting of the enlarged perforated spines, possibly by disassembling actin-myosin filaments that associate with the spine plasma membrane via phosphorylation and inactivation of myosin light chain kinase. In this case, PAK inhibition would directly lead to a greater proportion of larger perforated spines in the transgenic mice (Hayashi, 2004).

In a synapse, spine size has been shown to correlate with bouton size and the number of docked vesicles in the bouton. Consistent with these observations, it was found that the increased proportion of larger spines in transgenic cortical neurons correlates with an increased proportion of boutons with a larger pool of docked vesicles, while the density of docked vesicles did not differ between transgenic and wild-type neurons. Since the docked vesicle pool coincides with the readily-releasable pool (RRP) that determines the probability of neurotransmitter release, these results suggest a larger RRP and possibly a greater release probability per synapse in the transgenic neurons. A greater release probability per synapse together with a lower synapse number (due to the lower spine density) could explain the unaltered frequency of AMPAR-mediated mEPSC that was observed in the transgenic neurons. Since endogenous active PAK is nearly absent in the axons and boutons of mature neurons, the observed presynaptic alteration is likely a secondary effect of the postsynaptic alteration, perhaps occurring through communication between pre- and post-synaptic terminals via various molecules, including cell adhesion molecules and secreted proteins (Hayashi, 2004).

Taken together, the correlatory observations of altered distribution of synapse size and impaired bidirectional modifiability of synaptic strength in the cortex and deficient memory consolidation in dnPAK transgenic mice led to the proposal that the basal distribution of synapse size in the cortex must be within a range that is appropriate for bidirectional modifiability in order for a population of neurons to function as an effective memory network. Thus, in addition to structural plasticity of individual synapses, memory consolidation would depend on the basal synaptic structure in a population of synapses in the cortex. Future studies employing genetic or pharmacological perturbations will help to further understand how basal synaptic structure affects bidirectional modifiability and memory capability. Hopefully, such studies will provide mechanistic insight into the causes of cognitive dysfunction in mental retardation, since many of the genes that are implicated in mental retardation, including PAK3, are involved in the regulation of synaptic structure (Hayashi, 2004).

Targets of PAKs

see PAK-kinase Evolutionary homologs part 2/3 | part 3/3 |

PAK-kinase: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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