Interactive Fly, Drosophila



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Proteins interacting with Rac: Cdk5

Cyclin-dependent kinase 5 (Cdk5: see Drosophila Cdk5) and its neuron-specific regulator p35 are essential for neuronal migration and for the laminar configuration of the cerebral cortex. In addition, p35/Cdk5 kinase concentrates at the leading edges of axonal growth cones and regulates neurite outgrowth in cortical neurons in culture. The Rho family of small GTPases is implicated in a range of cellular functions, including cell migration and neurite outgrowth. The p35/Cdk5 kinase is shown to co-localize with Rac in neuronal growth cones. Furthermore, p35 associates directly with Rac in a GTP-dependent manner. Another Rac effector, Pak1 kinase, is also present in the Rac-p35/Cdk5 complexes and co-localizes with p35/Cdk5 and Rac at neuronal peripheries. The active p35/Cdk5 kinase causes Pak1 hyperphosphorylation in a Rac-dependent manner, which results in down-regulation of Pak1 kinase activity. Because the Rho family of GTPases and the Pak kinases are implicated in actin polymerization, the modification of Pak1, imposed by the p35/Cdk5 kinase, is likely to have an impact on the dynamics of the reorganization of the actin cytoskeleton in neurons, thus promoting neuronal migration and neurite outgrowth (Nikolic, 1998).

Cultures of cerebellar macroneurons were used to study the pattern of expression, subcellular localization, and function of the neuronal cdk5 activator p35 during laminin-enhanced axonal growth. The results obtained indicate that laminin, an extracellular matrix molecule capable of selectively stimulating axonal extension and promoting MAP1B phosphorylation at a proline-directed protein kinase epitope, selectively stimulates p35 expression, increases its association with the subcortical cytoskeleton, and accelerates its redistribution to the axonal growth cones. In addition, suppression of p35, (but not of a highly related isoform designated as p39), by antisense oligonucleotide treatment selectively reduces cdk5 activity, laminin-enhanced axonal elongation, and MAP1b phosphorylation. Taken collectively, the present results suggest that cdk5/p35 may serve as an important regulatory linking agent between environmental signals (e.g., laminin) and constituents of the intracellular machinery (e.g., MAP1B) involved in axonal elongation (Paglini, 1998).

Phosphorylation of tau (a heat-stable neuron-specific microtubule-associated protein) by cdk5 is stimulated in the presence of microtubules (MTs). This stimulation is due to an increased phosphorylation rate but there is no increase in the total amount of phosphorylation. Two-dimensional phosphopeptide map analysis shows that MTs stimulate phosphorylation of a specific peptide. Using Western blotting with antibodies that recognize phosphorylation-dependent epitopes within tau, the phosphorylation sites stimulated by the presence of MTs were found to be Ser202 and Thr205 (numbered according to the human tau isoform containing 441 residues). MT-dependent phosphorylation at Thr205 is observed in situ in rat cerebrum primary cultured neurons. Stimulated phosphorylation at Ser202 and Thr205 decreases the MT-nucleation activity of tau, which is in contrast to MT-independent phosphorylation at Ser235 and Ser404 (Wada, 1998).

Cyclin-dependent kinase 5 (Cdk5) was originally isolated by its close homology to the human CDC2 gene, which is a key regulator of cell cycle progression. However, unlike other Cdks, the activity of Cdk5 is required in post-mitotic neurons. The neuronal-specific p35 protein, which shares no homology to cyclins, was identified by virtue of its association with and activation of Cdk5. Gene targeting studies in mice have shown that the p35/Cdk5 kinase is required for the proper neuronal migration and development of the mammalian cortex. The regulation of the p35/Cdk5 kinase has been investigated. p35, the activator of Cdk5, is a short-lived protein with a half-life (t1/2) of 20 to 30 min. Specific proteasome inhibitors such as lactacystin greatly stabilize p35 in vivo. Ubiquitination of p35 can be readily demonstrated in vitro and in vivo. Inhibition of Cdk5 activity by a specific Cdk inhibitor, roscovitine, or by overexpression of a dominant negative mutant of Cdk5 increases the stability of p35 by 2- to 3-fold. Furthermore, phosphorylation mutants of p35 also stabilize p35 2- to 3-fold. Together, these observations demonstrate that the p35/Cdk5 kinase can be subject to rapid turnover in vivo and suggest that phosphorylation of p35 upon Cdk5 kinase activation plays a autoregulatory role in p35 degradation mediated by ubiquitin-mediated proteolysis (Patrick, 1998).

Recent work has shown that high molecular weight neurofilament (NF) proteins are phosphorylated in their carboxy-terminal tail portion by the enzyme cyclin-dependent kinase 5 (CDK-5). The tail domain of neurofilaments contains 52 tripeptide repeats, namely, Lys-Ser-Pro, which mainly exist as KSPXK and KSPXXX motifs (X = amino acid). CDK-5 specifically phosphorylates the serine residues within the KSPXK sites. The structural basis for this type of substrate selectivity was probed by studying the conformation of synthetic peptides containing either KSPXK or KSPXXX repeats designed from native neurofilament sequences. Synthetic peptides with KSPXK repeats are phosphorylated on serine with a recombinant CDK-5/p25 complex, whereas those with KSPXXX repeats are unreactive in this system. Circular dichroism (CD) studies in 50% TFE/H2O reveal a predominantly helical conformation for the KSPXXX-containing peptides, whereas the CD spectra for KSPXK-containing peptides indicates the presence of a high population of extended structures in water and 50% TFE solutions. However, detailed NMR analysis of one such peptide which includes two such KSPXK repeats, suggests a turn-like conformation encompassing the first KSPXK repeat. Restrained molecular dynamics calculations yield an unusually stable, folded structure with a double S-like bend incorporating the central residues of the peptide. The data suggest that a transient reverse turn or loop-type structure may be a requirement for CDK-5-promoted phosphate transfer to neurofilament-specific peptide segments (Sharma, 1998).

The ubiquitously expressed cyclin-dependent kinase 5 (cdk5) is essential for brain development. Bioactivation of cdk5 in the brain requires the presence of one of two related regulatory subunits, p35 and p39. Since either protein alone can activate cdk5, the significance of their coexistence as cdk5 kinase activators is unclear. To determine whether the two activators are expressed in different cells throughout the nervous system and during development, the tissue distributions of cdk5, p35, and p39 mRNAs in the rat were compared using in situ hybridization. In the adult rat, expression levels of p35 mRNA are generally higher in the brain than in the spinal cord, while the converse is observed for p39 mRNA. During neurogenesis, both p35 and p39 transcripts can be detected as early as embryonic day 12 (E12) in the marginal zone, but are absent from the ventricular zone, which may restrict cdk5 activation to the postmitotic neural cells in the developing brain. The expression levels of p35 and p39 mRNAs in the marginal zone increase by E15 and E17, paralleling the neurogenetic timetable. One exception is in the rostral forebrain, where p35 mRNA expression levels are high, suggesting that p35 may be the major activator for cdk5 during telencephalic morphogenesis. A significant level of p35 mRNA is present in the myotome at E12 and p35 expression persists in the premuscle mass and mature musculature at later stages, suggesting that p35 may also activate cdk5 during myogenesis (Zheng, 1998).

The Munc-18/syntaxin 1A complex has been postulated to act as a negative control on the regulated exocytotic process because its formation blocks the interaction of syntaxin with vesicle SNARE proteins. However, the formation of this complex is simultaneously essential for the final stages of secretion as evidenced by the necessity of Munc-18's homologs in Saccharomyces cerevisiae (Sec1p), Drosophila (ROP), and Caenorhabditis elegans (Unc-18) for proper secretion in these organisms. As such, any event that regulates the interaction of these two proteins is important for the control of secretion. One candidate for such regulation is cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division cycle kinases that has recently been copurified with Munc-18 from rat brain. The present study shows that Cdk5 bound to its neural specific activator p35 not only binds to Munc-18 but utilizes it as a substrate for phosphorylation. Furthermore, it is demonstrated that Munc-18, when it has been phosphorylated by Cdk5, has a significantly reduced affinity for syntaxin 1A. Cdk5 can also bind to syntaxin 1A and a complex of Cdk5, p35, Munc-18, and syntaxin 1A can be fashioned in the absence of ATP and promptly disassembled upon the addition of ATP. These results suggest a model in which p35-activated Cdk5 becomes localized to the Munc-18/syntaxin 1A complex by its affinity for both proteins so that it may phosphorylate Munc-18 and thus permit the positive interaction of syntaxin 1A with upstream protein effectors of the secretory mechanism (Shuang, 1998).

Cdk5 exists in brain extracts in multiple forms, one of which is a macromolecular protein complex comprising Cdk5, neuron-specific Cdk5 activator p35nck5a and other protein components. The yeast two-hybrid system was employed to identify p35nck5a-interacting proteins from a human brain cDNA library. One of the isolated clones encodes a fragment of glial fibrillary acidic protein, which is a glial-specific protein. Sequence alignment reveals significant homology between the p35nck5a-binding fragment of glial fibrillary acidic protein and corresponding regions in neurofilaments. The association between p35nck5a and neurofilament medium molecular weight subunit (NF-M) was confirmed by both the yeast two-hybrid assay and direct binding of the bacteria-expressed proteins. The p35nck5a binding site on NF-M was mapped to a carboxyl-terminal region of the rod domain, in close proximity to the putative Cdk5 phosphorylation sites in NF-M. A region immediately amino-terminal to the kinase-activating domain in p35nck5a is required for its binding with NF-M. In in vitro binding assays, NF-M binds both monomeric p35nck5a and the Cdk5/p35nck5a complex. The binding of NF-M has no effect on the kinase activity of Cdk5/p35nck5a (Qi, 1998).

The role of cyclin-dependent kinases in cell death has been investigated and the expression of cyclin-dependent kinase 5 (Cdk5) is found to be associated with apoptotic cell death in both adult and embryonic tissues. By double labeling immunohistochemistry and confocal microscopy, the expression of Cdk5 was specifically associated with dying cells. The association of Cdks with cell death is unique to Cdk5, since this association is not found with the other Cdks (Cdk 1-8) and cell death. The differential increase in Cdk5 expression is at the level of protein only, and no differences can be detected at the level of mRNA. Using the limbs of mutant mice detective in the pattern of interdigital cell death and limbs with increased interdigital cell death as a result of retinoic acid treatment, the specificity of Cdk5 protein expression in dying cells was confirmed. To investigate the regulation of Cdk5 during cell death, the expression of a regulatory protein of Cdk5, p35, was examined. p35 was found to be expressed in the dying cells as well. Similar to Cdk5, there is also no specific differential expression of the p35 mRNA in dying cells. These results suggest a role for Cdk5 and p35 proteins in cell death. This protein complex may function in the rearrangement of the cytoskeleton during apoptosis (Ahuja, 1998).

Cyclin-dependent kinase-5 (cdk-5) is a serine/threonine kinase that displays neurone-specific activity. Experimental manipulation of cdk-5 expression in neurons has shown that cdk-5 is essential for proper development of the nervous system and, in particular, for outgrowth of neurites. Such observations suggest that cdk-5 activity must be tightly controlled during development of the nervous system. To identify possible regulators of cdk-5, the yeast two-hybrid system was used to search for proteins that interact with cdk-5. In two independent yeast transformation events, cyclin D2 interacted with cdk-5. Immunoprecipitation experiments confirm that cyclin D2 and cdk-5 interact in mammalian cells. Cyclin D2 did not activate cdk-5 as assayed using three different substrates, which was in contrast to a known cdk-5 activator, p35. However, cyclin D2 expression leads to a decrease in cdk-5/p35 activity in transfected cells. As cyclin D2 and cdk-5 are known to share overlapping patterns of expression during development of the CNS, the results presented here suggest a role for cyclin D2 in modulating cdk-5 activity in postmitotic developing neurons (Guidato, 1998).

Rac interacts with a protein complex involved in the generation of reactive oxygen species

Phagocytes are important mediators of innate immunity through the generation of reactive oxygen species (ROS) that are capable of killing invading microorganisms. The source of these oxygen species is a superoxide anion (O2-) produced by the NADPH oxidase (the respiratory burst), a large, membrane-associated enzyme complex. In humans, the NADPH oxidase constitutes the primary defense mechanism against microbial infection, and mutations in any of the enzyme components can result in chronic granulomatous disease (CGD) in which patients suffer from severe bacterial and fungal infections due to defects in superoxide production. Since these highly reactive chemical species may also cause severe tissue damage and induce inflammatory responses, the respiratory burst must be tightly regulated. This is achieved by maintaining the oxidase in a dormant, inactive state in which the component proteins are partitioned between the cytosol and the cell membrane (Lapouge, 2000 and references therein).

The NADPH oxidase consists of at least six subunits: four cytosolic proteins [Rac, p40phox, p47phox and p67phox] together with two membrane-bound components [gp91phox and p22phox], which form a heterodimeric flavocytochrome, known as cytochrome b558. In resting cells, p40phox, p47phox, and p67phox exist in a cytoplasmic complex, which, upon activation, translocates to the membrane and associates with cytochrome b558 to form the active enzyme. Each of these proteins contains SH3 domains, which bind to proline-rich sequences present in p22phox, p47phox, and p67phox. The pairwise protein-protein interactions in these complexes have been extensively investigated, but the intra- and inter-molecular rearrangements that take place during activation and translocation to the membrane are still poorly understood (Lapouge, 2000 and references therein).

A crucial step in the assembly and the activation of the NADPH oxidase is the binding of the small GTPase Rac to p67phox. Inactive, GDP-bound Rac exists as a cytosolic complex with RhoGDI (Rho guanine nucleotide dissociation inhibitor) from which it dissociates upon activation to translocate to the plasma membrane, independently of the other cytosolic components. The interaction of Rac with p67phox is strictly GTP dependent, and much effort has been put into defining the regions in Rac and p67phox that are involved in complex formation (Lapouge, 2000 and references therein).

The region in p67phox mediating the interaction with Rac has been mapped to the N-terminal 200 amino acid residues, which have been predicted to contain four TPR (tetratrico-peptide repeat) motifs. TPR motifs are degenerate 34 amino acid repeats that are present in a variety of organisms, ranging from bacteria to humans. TPR motifs often occur in tandem arrays and mediate a range of protein-protein and possibly protein-lipid interactions. In fact, many TPR motif-containing proteins may act as scaffolds for the assembly of multiprotein complexes such as the anaphase promoting complex (APC) or the peroxisomal import receptor complex (Lapouge, 2000 and references therein).

Rac belongs to the Rho family of small GTPases, which are involved in a large variety of cellular processes. Like all small GTPases, Rac is active in its GTP-bound form and able to interact with a host of downstream effectors to induce specific cellular responses. These downstream effectors can be roughly devided into two groups, those specific for Rac and/or Cdc42 and those specific for Rho. A subset of Rac/Cdc42 effectors share a common motif, designated the CRIB motif (Cdc42/Rac interactive binding), which consists of a minimal region of 16 amino acids and is present in PAK (p21-activated kinase), ACK (activated Cdc42-associated kinase), and WASP (Wiskott-Aldrich syndrome protein). However, many more effectors specific for Rac and/or Cdc42 have been identified that lack the CRIB domain and share no obvious sequence homology in their GTPase binding domain. The recently solved structures of Cdc42 bound to CRIB domain containing peptides derived from ACK, WASP, and PAK reveal how CRIB domain effectors recognize their respective GTPase. However, no structural information has been available for complexes with Rac/Cdc42 specific effectors that do not contain a CRIB domain (Lapouge, 2000 and references therein).

The crystal structure of dominantly active Rac-Q61L-GTP bound to the TPR domain of p67phox is reported in this study along with biochemical analyses of the interaction. The structure reveals a completely different set of GTPase/effector interactions to those observed in CRIB domain binding and explains the observed biological specificity of p67phox for Rac. Complex formation is largely mediated by an insertion between two TPR motifs, suggesting an unsuspected versatility of TPR domains in target recognition and in their more general role as scaffolds for the assembly of multiprotein complexes. The Rac/p67phox complex involves a different mode of TPR domain-mediated protein-protein interaction from those previously described and suggests that TPR motifs may be used in different ways to create binding surfaces for the assembly of multiprotein complexes (Lapouge, 2000).

The Rac/p67TPR protein-protein interface is formed by residues that are highly conserved between Rac and Cdc42, yet Cdc42 shows no detectable binding. Studies employing point mutations and Rac/Cdc42 chimeras have suggested that Ala-27 and Gly-30 alone account for the specificity of the Rac/p67phox interaction. The crystal structure straightforwardly shows that Gly-30 is the only residue not conserved between Rac and Cdc42 that is present at the protein-protein interface. Ala-27 is not directly involved in the interface, but the structure suggests that substitution of this residue with Lys, which is present in Cdc42, could lead to a steric clash of its side chain with the beta hairpin insertion of p67phox. To directly examine the contribution of Ala-27 and Gly-30 to the specificity of the Rac/p67phox complex, the corresponding mutations were introduced into Cdc42 and binding to p67TPR was measured. In combination, these mutations result in a protein that binds to p67TPR with an affinity of 6 ┬ÁM, only about 2-fold lower than wild-type Rac1. Introduction of the corresponding residues from Cdc42 into Rac results in a protein that is unable to bind to p67TPR. These data confirm that specificity of p67phox for Rac is almost exclusively conferred by these two amino acids and that the contribution of other amino acids in the interface is purely to the stability of this complex (Lapouge, 2000).

Other signaling upstream of Rac

Netrins are a family of secreted proteins that guide the migration of cells and axonal growth cones during development. DCC (deleted in colorectal cancer) is a receptor for netrin-1 implicated in mediating these responses. DCC interacts constitutively with the SH3/SH2 adaptor Nck in commissural neurons. This interaction is direct and requires the SH3 but not SH2 domains of Nck-1. Moreover, both DCC and Nck-1 associate with the actin cytoskeleton, and this association is mediated by DCC. A dominant negative Nck-1 inhibits the ability of DCC to induce neurite outgrowth in N1E-115 cells and to activate Rac1 in fibroblasts in response to netrin-1. These studies provide evidence for an important role of mammalian Nck-1 in a novel signaling pathway from an extracellular guidance cue to changes in the actin-based cytoskeleton responsible for axonal guidance (Li, 2002a).

Netrins are chemotropic guidance cues that attract or repel growing axons during development. DCC (deleted in colorectal cancer), a transmembrane protein that is a receptor for netrin-1, is implicated in mediating both responses. However, the mechanism by which this is achieved remains unclear. This study reports that Rho GTPases are required for embryonic spinal commissural axon outgrowth induced by netrin-1. Using N1E-115 neuroblastoma cells, it was found that both Rac1 and Cdc42 activities are required for DCC-induced neurite outgrowth. In contrast, down-regulation of RhoA and its effector Rho kinase stimulates the ability of DCC to induce neurite outgrowth. In Swiss 3T3 fibroblasts, DCC was found to trigger actin reorganization through activation of Rac1 but not Cdc42 or RhoA. Stimulation of DCC receptors with netrin-1 results in a 4-fold increase in Rac1 activation. These results implicate the small GTPases Rac1, Cdc42, and RhoA as essential components that participate in signaling the response of axons to netrin-1 during neural development (Li, 2002b).

Cell polarization and migration in response to chemokines is essential for proper development of the immune system and activation of immune responses. Recent studies of chemokine signaling have revealed a critical role for PI3-Kinase, which is required for polarized membrane association of pleckstrin homology (PH) domain-containing proteins and activation of Rho family GTPases that are essential for cell polarization and actin reorganization. Additional data argue that tyrosine kinases are also important for chemokine-induced Rac activation. However, how and which kinases participate in these pathways remain unclear. The Tec kinases Itk and Rlk play an important role in chemokine signaling in T lymphocytes. Chemokine stimulation induces transient membrane association of Itk and phosphorylation of both Itk and Rlk, and purified T cells from Rlk-/-Itk-/- mice exhibits defective migration to multiple chemokines in vitro and decreased homing to lymph nodes upon transfer to wt mice. SDF-1alpha is a chemokine that acts via the broadly expressed receptor CXCR4. Expression of a dominant-negative Itk impairs SDF-1alpha-induced migration, cell polarization, and activation of Rac and Cdc42. Thus, Tec kinases are critical components of signaling pathways required for actin polarization downstream from both antigen and chemokine receptors in T cells (Takesono, 2004).

To determine whether chemokine stimulation activates Tec kinases, the effects were examined of SDF-1alpha on phosphorylation of endogenous Itk, the major Tec kinase expressed in the Jurkat T cell line. In this cell line, Itk is constitutively localized at the plasma membrane due to a deficiency of the lipid phosphatase PTEN. Exposure of Jurkat cells to SDF-1alpha leads to a rapid increase in Itk tyrosine phosphorylation. Consistent with the known role of Lck in activation of Itk and previous reports that Lck is important for chemokine-induced migration of Jurkat cells, inhibition of Src family kinases with PP2 prevents Itk phosphorylation. Furthermore, examination of a transfected murine Rlk construct revealed that SDF-1alpha also induces tyrosine phosphorylation of Rlk. Thus, chemokines appear to stimulate phosphorylation of both Itk and Rlk (Takesono, 2004).

Itk has been shown to be required for TCR-induced actin polarization and the activation of Cdc42 and its downstream effector WASP, a critical regulator of the actin cytoskeleton that contributes to lymphocyte chemotaxis. To determine whether similar defects are observed downstream of chemokine receptor signaling, polarization was examined of Jurkat cells that were bound to beads coated with either fibronectin or fibronectin and SDF-1alpha. In the absence of SDF-1alpha or in the presence of SDF-1alpha at 0°C, cells bound the fibronectin-coated beads yet retained a spherical morphology. Stimulation of wt Jurkat cells or Jurkat cells expressing GFP with beads coated with fibronectin and SDF-1? at 37°C led to rapid changes in cell morphology, with increased F-actin accumulation associated with engulfment of the beads. However, cells expressing ItkKD-GFP failed to elongate and exhibited polarized actin accumulation upon SDF-1alpha stimulation. Moreover, ItkKD-GFP cells also showed decreased actin polymerization in response to SDF-1alpha as assessed by flow cytometry of fluorescently labeled phalloidin binding (Takesono, 2004).

To determine whether Itk also regulates chemokine-induced activation of Rho family GTPases, a GST-PAK-CRIB pull-down assay was used to isolate active, GTP bound Cdc42 and Rac. Stimulation of GFP-expressing Jurkat cells with SDF-1alpha led to the rapid activation of both Rac and Cdc42. However, Jurkat cells expressing ItkKD-GFP showed impaired SDF-1alpha-induced Rac and Cdc42 activation. In contrast, activation of Erk in response to SDF-1alpha was only minimally affected in the ItkKD-GFP cells, suggesting that some chemokine-mediated signaling pathways are intact in these cells (Takesono, 2004).

Cadherin adhesion molecules are key determinants of morphogenesis and tissue architecture. Nevertheless, the molecular mechanisms responsible for the morphogenetic contributions of cadherins remain poorly understood in vivo. Besides supporting cell-cell adhesion, cadherins can affect a wide range of cellular functions that include activation of cell signalling pathways, regulation of the cytoskeleton and control of cell polarity. To determine the role of E-cadherin in stratified epithelium of the epidermis, its gene was conditionally inactivated in mice. Loss of E-cadherin in the epidermis in vivo results in perinatal death of mice due to the inability to retain a functional epidermal water barrier. Absence of E-cadherin leads to improper localization of key tight junctional proteins, resulting in permeable tight junctions and thus altered epidermal resistance. In addition, both Rac and activated atypical PKC, crucial for tight junction formation, are mislocalized. Surprisingly, the results indicate that E-cadherin is specifically required for tight junction (but not desmosome) formation and this appears to involve signalling rather than cell contact formation (Tunggal, 2005).

The non-receptor tyrosine kinase Abl participates in receptor tyrosine kinase (RTK)-induced actin cytoskeleton remodelling, a signalling pathway in which the function of Rac is pivotal. More importantly, the activity of Rac is indispensable for the leukaemogenic ability of the BCR-Abl oncoprotein. Thus, Rac might function downstream of Abl and be activated by it. This study elucidates the molecular mechanisms through which Abl signals to Rac in RTK-activated pathways. Sos-1, a dual guanine nucleotide-exchange factor (GEF), is phosphorylated on tyrosine, after activation of RTKs, in an Abl-dependent manner. Sos-1 and Abl interact in vivo, and Abl-induced tyrosine phosphorylation of Sos-1 is sufficient to elicit its Rac-GEF activity in vitro. Genetic or pharmacological interference with Abl (and the related kinase Arg) results in a marked decrease in Rac activation induced by physiological doses of growth factors. Thus, these data identify the molecular connections of a pathway RTKs-Abl-Sos-1-Rac that is involved in signal transduction and actin remodelling (Sini, 2004).

Proteins at cell-extracellular matrix adhesions (e.g. focal adhesions) are crucially involved in regulation of cell morphology and survival. CH-ILKBP/actopaxin/alpha-parvin and affixin/beta-parvin (abbreviated as alpha- and beta-parvin, respectively; see Drosophila Parvin), two structurally closely related integrin-linked kinase (ILK)-binding focal adhesion proteins, are co-expressed in human cells. Depletion of alpha-parvin dramatically increases the level of beta-parvin, suggesting that beta-parvin is negatively regulated by alpha-parvin in human cells. Loss of PINCH-1 (Drosophila homolog: Steamer duck) or ILK, to which alpha- and beta-parvin bind, significantly reduces the activation of Rac, a key signaling event that controls lamellipodium formation and cell spreading. It was surprising to find that loss of alpha-parvin, but not that of beta-parvin, markedly stimulates Rac activation and enhances lamellipodium formation. Overexpression of beta-parvin, however, is insufficient for stimulation of Rac activation or lamellipodium formation, although it is sufficient for promotion of apoptosis, another important cellular process that is regulated by PINCH-1, ILK, and alpha-parvin. In addition, the interactions of ILK with alpha- and beta-parvin are mutually exclusive. Overexpression of beta-parvin or its CH(2) fragment, but not a CH(2) deletion mutant, inhibited the ILK-alpha-parvin complex formation. Finally, evidence is provided suggesting that inhibition of the ILK-alpha-parvin complex is sufficient, although not necessary, for promotion of apoptosis. These results identify Rac as a downstream target of PINCH-1, ILK, and parvin. Furthermore, they demonstrate that alpha- and beta-parvins play distinct roles in mammalian cells and suggest that the formation of the ILK-alpha-parvin complex is crucial for protection of cells from apoptosis (Zhang, 2004).

Neuronal activity augments maturation of mushroom-shaped spines to form excitatory synapses, thereby strengthening synaptic transmission. A Ca2+-signaling pathway downstream of the NMDA receptor has been delineated that stimulates calmodulin-dependent kinase kinase (CaMKK) and CaMKI to promote formation of spines and synapses in hippocampal neurons. CaMKK and CaMKI form a multiprotein signaling complex with the guanine nucleotide exchange factor (GEF) βPIX and GIT1 that is localized in spines. CaMKI-mediated phosphorylation of Ser516 in βPIX enhances its GEF activity, resulting in activation of Rac1, an established enhancer of spinogenesis. Suppression of CaMKK or CaMKI by pharmacological inhibitors, dominant-negative (dn) constructs and siRNAs, as well as expression of the βPIX Ser516Ala mutant, decreases spine formation and mEPSC frequency. Constitutively-active Pak1, a downstream effector of Rac1, rescues spine inhibition by dnCaMKI or βPIX S516A. This activity-dependent signaling pathway can promote synapse formation during neuronal development and in structural plasticity (Saneyoshi, 2008).

Microglia Dysfunction Caused by the Loss of Rhoa Disrupts Neuronal Physiology and Leads to Neurodegeneration

Nervous tissue homeostasis requires the regulation of microglia activity. Using conditional gene targeting in mice, this study demonstrates that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation, producing a neurological phenotype (including synapse and neuron loss, impairment of long-term potentiation [LTP], formation of beta-amyloid plaques, and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src (see Drosophila Src64) activation and Src-mediated tumor necrosis factor (TNF) production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. It was also found that the Rhoa/Src signaling pathway is disrupted in microglia of the APP/PS1 mouse model of Alzheimer disease and that low doses of Abeta oligomers trigger microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, these results indicate that disturbing Rho GTPase signaling in microglia can directly cause neurodegeneration (Socodato, 2020).

Table of contents

Rac1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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