Cdc42 Binding Research Articles

A PDZ-kinase allosteric relay mediates Par complex regulator exchange.

The Par complex polarizes the plasma membrane of diverse animal cells using the catalytic activity of atypical Protein Kinase C (aPKC) to pattern substrates. Two upstream regulators of the Par complex, Cdc42 and Par-3, bind separately to the complex to influence its activity in different ways. Each regulator binds a distinct member of the complex, Cdc42 to Par-6 and Par-3 to aPKC, making it unclear how they influence one another's binding. Here we report the discovery that Par-3 binding to aPKC is regulated by aPKC autoinhibition and link this regulation to Cdc42 and Par-3 exchange. The Par-6 PDZ domain activates aPKC binding to Par-3 via a novel interaction with the aPKC kinase domain. Cdc42 and Par-3 have opposite effects on the Par-6 PDZ-aPKC kinase interaction: while the Par-6 kinase domain interaction competes with Cdc42 binding to the complex, Par-3 binding is enhanced by the interaction. The differential effect of Par-3 and Cdc42 on the Par-6 PDZ interaction with the aPKC kinase domain forms an allosteric relay that connects their binding sites and is responsible for the negative cooperativity that underlies Par complex polarization and activity.

Bayesian mapping of protein kinases to vasopressin-regulated phosphorylation sites in renal collecting duct.

Vasopressin controls water permeability in the renal collecting duct by regulating the water channel protein, aquaporin-2 (AQP2). Phosphoproteomic studies have identified multiple proteins that undergo phosphorylation changes in response to vasopressin. The kinases responsible for the phosphorylation of most of these sites have not been identified. Here, we use large-scale Bayesian data integration to predict the responsible kinases for 51 phosphoproteomically identified vasopressin-regulated phosphorylation sites in the renal collecting duct. To do this, we applied Bayes' rule to rank the 515 known mammalian protein kinases for each site. Bayes' rule was applied recursively to integrate each of the seven independent datasets, each time using the posterior probability vector of a given step as the prior probability vector of the next step. In total, 30 of the 33 phosphorylation sites that increase with vasopressin were predicted to be phosphorylated by protein kinase A (PKA) catalytic subunit-α, consistent with prior studies implicating PKA in vasopressin signaling. Eighteen of the vasopressin-regulated phosphorylation sites were decreased in response to vasopressin and all but three of these sites were predicted to be targets of extracellular signal-regulated kinases, ERK1 and ERK2. This result implies that ERK1 and ERK2 are inhibited in response to vasopressin V2 receptor occupation, secondary to PKA activation. The six phosphorylation sites not predicted to be phosphorylated by PKA or ERK1/2 are potential targets of other protein kinases previously implicated in aquaporin-2 regulation, including cyclin-dependent kinase 18 (CDK18), calmodulin-dependent kinase 2δ (CAMK2D), AMP-activated kinase catalytic subunit-α-1 (PRKAA1) and CDC42 binding protein kinase β (CDC42BPB).NEW & NOTEWORTHY Vasopressin regulates water transport in the renal collecting duct in part through phosphorylation or dephosphorylation of proteins that regulate aquaporin-2. Prior studies have identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. This study uses Bayesian data integration techniques to combine information from multiple prior proteomics and transcriptomics studies to predict the protein kinases that phosphorylate the 51 sites. Most of the regulated sites were predicted to be phosphorylated by protein kinase A or ERK1/ERK2.

The Chlamydia pneumoniae effector SemD exploits its host’s endocytic machinery by structural and functional mimicry

To enter epithelial cells, the obligate intracellular pathogen Chlamydia pneumoniae secretes early effector proteins, which bind to and modulate the host-cell’s plasma membrane and recruit several pivotal endocytic host proteins. Here, we present the high-resolution structure of an entry-related chlamydial effector protein, SemD. Co-crystallisation of SemD with its host binding partners demonstrates that SemD co-opts the Cdc42 binding site to activate the actin cytoskeleton regulator N-WASP, making active, GTP-bound Cdc42 superfluous. While SemD binds N-WASP much more strongly than Cdc42 does, it does not bind the Cdc42 effector protein FMNL2, indicating effector protein specificity. Furthermore, by identifying flexible and structured domains, we show that SemD can simultaneously interact with the membrane, the endocytic protein SNX9, and N-WASP. Here, we show at the structural level how a single effector protein can hijack central components of the host’s endocytic system for efficient internalization.

Characterization of Anticancer Effects of the Analogs of DJ4, a Novel Selective Inhibitor of ROCK and MRCK Kinases.

The Rho associated coiled-coil containing protein kinase (ROCK1 and ROCK2) and myotonic dystrophy-related Cdc-42 binding kinases (MRCKα and MRCKβ) are critical regulators of cell proliferation and cell plasticity, a process intimately involved in cancer cell migration and invasion. Previously, we reported the discovery of a novel small molecule (DJ4) selective multi-kinase inhibitor of ROCK1/2 and MRCKα/β. Herein, we further characterized the anti-proliferative and apoptotic effects of DJ4 in non-small cell lung cancer and triple-negative breast cancer cells. To further optimize the ROCK/MRCK inhibitory potency of DJ4, we generated a library of 27 analogs. Among the various structural modifications, we identified four additional active analogs with enhanced ROCK/MRCK inhibitory potency. The anti-proliferative and cell cycle inhibitory effects of the active analogs were examined in non-small cell lung cancer, breast cancer, and melanoma cell lines. The anti-proliferative effectiveness of DJ4 and the active analogs was further demonstrated against a wide array of cancer cell types using the NCI-60 human cancer cell line panel. Lastly, these new analogs were tested for anti-migratory effects in highly invasive MDA-MB-231 breast cancer cells. Together, our results demonstrate that selective inhibitors of ROCK1/2 (DJE4, DJ-Allyl) inhibited cell proliferation and induced cell cycle arrest at G2/M but were less effective in cell death induction compared with dual ROCK1/2 and MRCKα/β (DJ4 and DJ110).

Why Does Synergistic Activation of WASP, but Not N-WASP, by Cdc42 and PIP2 Require Cdc42 Prenylation?

Human WASP and N-WASP are homologous proteins that require the binding of multiple regulators, including the acidic lipid PIP2 and the small GTPase Cdc42, to relieve autoinhibition before they can stimulate the initiation of actin polymerization. Autoinhibition involves intramolecular binding of the C-terminal acidic and central motifs to an upstream basic region and GTPase binding domain. Little is known about how a single intrinsically disordered protein, WASP or N-WASP, binds multiple regulators to achieve full activation. Here we used molecular dynamics simulations to characterize the binding of WASP and N-WASP with PIP2 and Cdc42. In the absence of Cdc42, both WASP and N-WASP strongly associate with PIP2-containing membranes, through their basic region and also possibly through a tail portion of the N-terminal WH1 domain. The basic region also participates in Cdc42 binding, especially for WASP; consequently Cdc42 binding significantly compromises the ability of the basic region in WASP, but not N-WASP, to bind PIP2. PIP2 binding to the WASP basic region is restored only when Cdc42 is prenylated at the C-terminus and tethered to the membrane. This distinction in the activation of WASP and N-WASP may contribute to their different functional roles.

Full activation of WASP, not N-WASP, by Cdc42 and PIP2 requires Cdc42 prenylation.

WASP and N-WASP are homologous proteins that require the binding of multiple regulators, including the acidic lipid PIP2 and the small GPTase Cdc42, to relieve auto-inhibition before they can stimulate the initiation of actin polymerization. Little is known about how a single intrinsically disordered protein, WASP or N-WASP, binds multiple regulators to achieve full activation. Here we used molecular dynamics simulations to characterize the binding of WASP and N-WASP with PIP2 and Cdc42. In the absence of Cdc42, both WASP and N-WASP strongly associate with PIP2-containing membranes, through their basic region and also possibly through a tail portion of the upstream WH1 domain. The basic region also participates in Cd42 binding, especially for WASP; consequently, Cdc42 binding significantly compromises the ability of WASP, but not N-WASP, to bind PIP2. PIP2 binding of WASP is restored only when Cdc42 is prenylated at the C-terminus and tethered to the membrane. This distinction in the full activation of WASP and N-WASP may contribute to their different function roles.

Discovery of small molecule inhibitors that effectively disrupt IQGAP1-Cdc42 interaction in breast cancer cells

The small GTPase Cdc42 is an integral component of the cytoskeleton, and its dysregulation leads to pathophysiological conditions, such as cancer. Binding of Cdc42 to the scaffold protein IQGAP1 stabilizes Cdc42 in its active form. The interaction between Cdc42 and IQGAP1 enhances migration and invasion of cancer cells. Disrupting this association could impair neoplastic progression and metastasis; however, no effective means to achieve this has been described. Here, we screened 78,500 compounds using a homogeneous time resolved fluorescence-based assay to identify small molecules that disrupt the binding of Cdc42 to IQGAP1. From the combined results of the validation assay and counter-screens, we selected 44 potent compounds for cell-based experiments. Immunoprecipitation and cell viability analysis rendered four lead compounds, namely NCGC00131308, NCGC00098561, MLS000332963 and NCGC00138812, three of which inhibited proliferation and migration of breast carcinoma cells. Microscale thermophoresis revealed that two compounds bind directly to Cdc42. One compound reduced the amount of active Cdc42 in cells and effectively impaired filopodia formation. Docking analysis provided plausible models of the compounds binding to the hydrophobic pocket adjacent to the GTP binding site of Cdc42. In conclusion, we identified small molecules that inhibit binding between Cdc42 and IQGAP1, which could potentially yield chemotherapeutic agents.

CDC42-IQGAP Interactions Scrutinized: New Insights into the Binding Properties of the GAP-Related Domain.

The IQ motif-containing GTPase-activating protein (IQGAP) family composes of three highly-related and evolutionarily conserved paralogs (IQGAP1, IQGAP2 and IQGAP3), which fine tune as scaffolding proteins numerous fundamental cellular processes. IQGAP1 is described as an effector of CDC42, although its effector function yet re-mains unclear. Biophysical, biochemical and molecular dynamic simulation studies have proposed that IQGAP RASGAP-related domains (GRDs) bind to the switch regions and the insert helix of CDC42 in a GTP-dependent manner. Our kinetic and equilibrium studies have shown that IQGAP1 GRD binds, in contrast to its C-terminal 794 amino acids (called C794), CDC42 in a nucleotide-independent manner indicating a binding outside the switch regions. To resolve this discrepancy and move beyond the one-sided view of GRD, we carried out affinity measurements and a systematic mutational analysis of the interfacing residues between GRD and CDC42 based on the crystal structure of the IQGAP2 GRD-CDC42Q61L GTP complex. We determined a 100-fold lower affinity of the GRD1 of IQGAP1 and of GRD2 of IQGAP2 for CDC42 mGppNHp in comparison to C794/C795 proteins. Moreover, partial and major mutation of CDC42 switch regions substantially affected C794/C795 binding but only a little GRD1 and remarkably not at all the GRD2 binding. However, we clearly showed that GRD2 contributes to the overall affinity of C795 by using a 11 amino acid mutated GRD variant. Furthermore, the GRD1 binding to the CDC42 was abolished using specific point mutations within the insert helix of CDC42 clearly supporting the notion that CDC42 binding site(s) of IQGAP GRD lies outside the switch regions among others in the insert helix. Collectively, this study provides further evidence for a mechanistic framework model that is based on a multi-step binding process, in which IQGAP GRD might act as a ‘scaffolding domain’ by binding CDC42 irrespective of its nucleotide-bound forms, followed by other IQGAP domains downstream of GRD that act as an effector domain and is in charge for a GTP-dependent interaction with CDC42.

A multiplex model in a Drosophilaidentifies novel gene‐environment interactions: A step towards personalized medicine

Epidemiological studies strongly support a role for environmental factors, particularly pesticide exposure, in the pathogenesis of Parkinson's disease (PD). Genetic risk factors, including penetrant single‐gene mutations and risk factors identified from genome‐wide associated studies (GWAS), also contribute to PD risk and progression, but available model systems are limited in their ability to interrogate gene‐environment crosstalk in vivo and at scale. Here we use Drosophila as a model system to understand complex gene‐environment interactions in PD. We developed a multiplex model in Drosophilain which we knocked down GWAS candidate genes in neurons in a robust new α‐synucleinopathy model and exposed the flies to environmental neurotoxicants. We identified an interaction between LRRK2, rotenone, and α‐synuclein. Expression of the disease‐causing Lrrk‐G2019S mutant in the presence of rotenone and alpha‐synuclein induced behavioral deficits and mitochondrial dysfunction. Further, super‐resolution microscopy analysis revealed that the interaction of LRRK2, α‐synuclein, and rotenone leads to hyperstabilization of the actin cytoskeleton. We have previously shown that LRRK2 has actin severing activity, and previous studies have implicated the GTPase domain in regulating actin severing. We next expressed a GTPase domain mutant, Lrrk‐Q1003H, designed to mimic the human protective mutant LRRK2‐R1398H. Interestingly, expression of the LRRK2 protective mutant attenuated behavioral deficits mediated by LRRK2‐rotenone‐synuclein interactions. Further, the expression of the protective mutant also attenuated the actin stabilization and mitochondrial deficits. Moreover, genetic analysis from a patient cohort who had previous pesticide exposure revealed that the patient's LRRK2‐R1398H mutation reduces the chance of developing PD. Since global actin severing may have unwanted side effects, we used a combination of forward genetic screen and proteomics to identify potential kinases that can be druggable targets. We identified Cdc42 binding protein kinase MRCKα, an actin‐binding protein, as a potential target. Genetic and pharmacological inhibition of MRCKα in Drosophila can modify the toxicity induced by the interaction among LRRK2, α‐synuclein, and rotenone by inhibiting actin hyperstabilization. Using our novel multiplex model in Drosophila, we have identified an interaction between LRRK2, α‐synuclein, and rotenone which is modulated by actin stabilization and mitochondrial dysfunction. We have further demonstrated that using this multiplex approach, we can study the mechanism of these interactions as well as identify novel drug targets for the interactions. Our findings have implications towards the development of a personalized approach for drug discovery and lead identification.

Gene Delivery of MRCKα Repairs the Alveolar‐Capillary Barrier and Rescues the Experimental Lipopolysaccharide‐Induced Acute Lung Injury

Rationale Acute Respiratory Distress Syndrome (ARDS) is a devastating condition which is characterized by the pulmonary accumulation of protein-rich edema fluids and insufficient gas exchange, leading to acute hypoxemic respiratory failure. During ARDS, the net fluid clearance is undermined not only due to the impaired alveolar fluid clearance (AFC) which osmotically drives water following the ion gradient out of alveoli, but also the disrupted alveolar-capillary barrier. The previous data from our lab indicated that electroporation-mediated gene delivery of the β1 subunit of the Na+, K+-ATPase into mouse lungs treats LPS induced acute lung injury (ALI), showing significantly decreased lung edema, and improved overall outcome of lung injury. Overexpression of β1 increased AFC in healthy and injured murine lungs, and more importantly, gene delivery of β1 restored pulmonary barrier function, demonstrated by upregulated tight junction (TJ) proteins, and decreased lung permeability, total protein and cellularity in BAL fluid. MRCKα (CDC42 binding protein kinase alpha) was identified by our lab as an interacting partner of the β1 subunit through mass spectrometry. MRCKα is a Rho GTPase effector kinase, regulating diverse cell behaviors, including junction formation. Our previous in vitro data indicate that MRCKα mediated β1’s upregulation of TJ proteins and epithelial barrier integrity. Overexpression of MRCKα alone sufficiently increased the trans-epithelial electrical resistance (TEER) in cell culture. Thus, it is intriguing to investigate whether MRCKα has the therapeutic potential to treat LPS induced ALI by restoring pulmonary barrier function in vivo. This study tested whether electroporation-mediated gene transfer of MRCKα could upregulate TJ protein and barrier function and treat LPS induced ALI in mice. Methods ALI was induced by LPS intratracheal administration and 24 hours later, plasmids encoding MRCKα, β1- Na+, K+-ATPase, or nothing (control) were delivered individually or in combination to mouse lungs by oropharyngeal aspiration and electroporation. Two days after gene delivery, various endpoint assays were performed to evaluate lung edema, permeability, inflammation and histological injury. In addition, AFC was performed on mice with MRCKα overexpression to evaluate the fluid clearance. Results Gene transfer of MRCKα alone did not enhance AFC, but alone or in combination with β1- Na+, K+-ATPase attenuated LPS-increased Wet/Dry ratio, lung leakage, and BAL cellularity and protein concentration, restored TJ protein expression, and improved overall outcome of lung injury. Conclusions Gene delivery ofMRCKα repair alveolar capillary barrier integrity without promoting ion transport, and could benefit the pre-injured lungs by reducing pulmonary edema, restoring lung barrier function and reducing inflammation. Moreover, the data also suggest that improving barrier function alone may be of equal or even more benefit than improving alveolar fluid clearance in order to treat ALI/ARDS.

A Novel Truncating Mutation in HOMER2 Causes Nonsyndromic Progressive DFNA68 Hearing Loss in a Spanish Family.

Nonsyndromic hereditary hearing loss is a common sensory defect in humans that is clinically and genetically highly heterogeneous. So far, 122 genes have been associated with this disorder and 50 of them have been linked to autosomal dominant (DFNA) forms like DFNA68, a rare subtype of hearing impairment caused by disruption of a stereociliary scaffolding protein (HOMER2) that is essential for normal hearing in humans and mice. In this study, we report a novel HOMER2 variant (c.832_836delCCTCA) identified in a Spanish family by using a custom NGS targeted gene panel (OTO-NGS-v2). This frameshift mutation produces a premature stop codon that may lead in the absence of NMD to a shorter variant (p.Pro278Alafs*10) that truncates HOMER2 at the CDC42 binding domain (CBD) of the coiled-coil structure, a region that is essential for protein multimerization and HOMER2-CDC42 interaction. c.832_836delCCTCA mutation is placed close to the previously identified c.840_840dup mutation found in a Chinese family that truncates the protein (p.Met281Hisfs*9) at the CBD. Functional assessment of the Chinese mutant revealed decreased protein stability, reduced ability to multimerize, and altered distribution pattern in transfected cells when compared with wild-type HOMER2. Interestingly, the Spanish and Chinese frameshift mutations might exert a similar effect at the protein level, leading to truncated mutants with the same Ct aberrant protein tail, thus suggesting that they can share a common mechanism of pathogenesis. Indeed, age-matched patients in both families display quite similar hearing loss phenotypes consisting of early-onset, moderate-to-profound progressive hearing loss. In summary, we have identified the third variant in HOMER2, which is the first one identified in the Spanish population, thus contributing to expanding the mutational spectrum of this gene in other populations, and also to clarifying the genotype–phenotype correlations of DFNA68 hearing loss.

Active and Inactive Cdc42 Differ in Their Insert Region Conformational Dynamics

Cell division control protein 42 homolog (Cdc42) protein, a Ras superfamily GTPase, regulates cellular activities, including cancer progression. Using all-atom molecular dynamics (MD) simulations and essential dynamic analysis, we investigated the structure and dynamics of the catalytic domains of GDP-bound (inactive) and GTP-bound (active) Cdc42 in solution. We discovered substantial differences in the dynamics of the inactive and active forms, particularly in the “insert region” (residues 122–135), which plays a role in Cdc42 activation and binding to effectors. The insert region has larger conformational flexibility in the GDP-bound Cdc42 than in the GTP-bound Cdc42. The G2 loop and switch I at the effector lobe of the catalytic domain exhibit large conformational changes in both the GDP- and the GTP-bound systems, but in the GTP-bound Cdc42, the switch I interactions with GTP are retained. Oncogenic mutations were identified in the Ras superfamily. In Cdc42, the G12V and Q61L mutations decrease the GTPase activity. We simulated these mutations in both GDP- and GTP-bound Cdc42. Although the overall structural organization is quite similar between the wild type and the mutants, there are small differences in the conformational dynamics, especially in the two switch regions. Taken together, the G12V and Q61L mutations may play a role similar to their K-Ras counterparts in nucleotide binding and activation. The conformational differences, which are mainly in the insert region and, to a lesser extent, in the switch regions flanking the nucleotide binding site, can shed light on binding and activation. We propose that the differences are due to a network of hydrogen bonds that gets disrupted when Cdc42 is bound to GDP, a disruption that does not exist in other Rho GTPases. The differences in the dynamics between the two Cdc42 states suggest that the inactive conformation has reduced ability to bind to effectors.

Phospho‐proteomic Analysis of Protease‐activated Receptor‐1 Biased Signaling Reveals Novel Modulators of Endothelial Barrier Function

Dysregulation of the vascular endothelium is a hallmark of inflammation and promotes barrier disruption, vascular leakage and tissue edema. Vascular endothelial dysfunction is mediated by vasoactive agents and inflammatory mediators, many of which signal through G protein‐coupled receptors (GPCRs). A key GPCR inflammatory mediator is thrombin, a procoagulant protease generated in response to vascular injury and inflammation. Thrombin activates protease‐activated receptor‐1 (PAR1) in endothelial cells to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC) is an anticoagulant protease that activates PAR1 through a distinct mechanism and promotes endothelial barrier stabilization. The ability of distinct tethered ligands formed through cleavage of PAR1 by different proteases results in stabilization of unique receptor active conformational states that promote opposite signaling effects is termed biased signaling. Despite progress in understanding PAR1 bias signaling in endothelial cells, the proteins and pathways by which thrombin and APC regulate the endothelial barrier integrity are largely unknown. Here, we report the global phosphoproteome of thrombin and APC signaling using tandem‐mass‐tag (TMT) mass spectrometry and computational analyses. Our results reveal unique temporal dynamics profiles of thrombin and APC signaling and the identification of multiple enriched phosphoproteome associated biological functions including microtubules, adherens junctions, actin and signaling, all key modulators of endothelial barrier permeability. We selected candidates that showed statistically significant changes in phosphorylation following thrombin or APC stimulation, and validated the function of four proteins including: afadin (AFDN), CDC42 binding protein kinase alpha (BPA), adducin 1 (ADD1), and GPCR interacting protein 1 (GIT1) using an siRNA‐targeted approach. Using target‐specific siRNAs to deplete candidate proteins in endothelial cells, we found that depletion of afadin and adducin had statistically significant effects on endothelial barrier permeability. Afadin depletion led to an increase in thrombin‐induced permeability, while adducin depletion virtually ablated thrombin‐stimulated permeability. Immunofluorescence microscopy imaging of morphological changes in F‐actin stress fiber formation supported these results and revealed corresponding changes in thrombin‐induced gap formation consistent with the observed changes in permeability. In addition, depletion of candidate proteins also modulated thrombin‐stimulated p38 pro‐inflammatory signaling and APC‐induced AKT cytoprotective signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 bias signaling in endothelial cells.Support or Funding InformationThis work was supported by NIH/NIGMS R01 GM116597 and R35 GM127121 (J.T.), UC Tobacco‐related Disease Research Program Predoctoral Fellowship (C.C.R), and NIH/NIGMS K12 GM068524 (O.M.I., J.D.L.)

Systems Genetics of Optic Nerve Axon Necrosis During Glaucoma.

In this study, we identify genomic regions that modulate the number of necrotic axons in optic nerves of a family of mice, some of which have severe glaucoma, and define a set of high priority positional candidate genes that modulate retinal ganglion cell (RGC) axonal degeneration. A large cohort of the BXD family were aged to greater than 13 months of age. Optic nerves from 74 strains and the DBA/2J (D2) parent were harvested, sectioned, and stained with p-phenylenediamine. Numbers of necrotic axons per optic nerve cross-section were counted from 1 to 10 replicates per genotype. Strain means and standard errors were uploaded into GeneNetwork 2 for mapping and systems genetics analyses (Trait 18614). The number of necrotic axons per nerve ranged from only a few hundred to more than 4,000. Using conventional interval mapping as well as linear mixed model mapping, we identified a single locus on chromosome 12 between 109 and 112.5 Mb with a likelihood ratio statistic (LRS) of ~18.5 (p genome-wide ~0.1). Axon necrosis is not linked to locations of major known glaucoma genes in this family, including Gpnmb, Tyrp1, Cdh11, Pou6f2, and Cacna2d1. This indicates that although these genes contribute to pigmentary dispersion or elevated IOP, none directly modulates axon necrosis. Of 156 positional candidates, eight genes—CDC42 binding protein kinase beta (Cdc42bpb); eukaryotic translation initiation factor 5 (Eif5); BCL2-associated athanogene 5 (Bag5); apoptogenic 1, mitochondrial (Apopt1); kinesin light chain 1 (Klc1); X-ray repair cross complementing 3 (Xrcc3); protein phosphatase 1, regulatory subunit 13B (Ppp1r13b); and transmembrane protein 179 (Tmem179)—passed stringent criteria and are high priority candidates. Several candidates are linked to mitochondria and/or axons, strengthening their plausible role as modulators of ON necrosis. Additional studies are required to validate and/or eliminate plausible candidates. Surprisingly, IOP and ON necrosis are inversely correlated across the BXD family in mice >13 months of age and these two traits share few genes among their top ocular and retinal correlates. These data suggest that the two traits are independently modulated or that a more complex and multifaceted approach is required to reveal their association.

WASP Restricts Active Rac to Maintain Cells' Front-Rear Polarization.

SummaryEfficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.

The subcellular localization of type I p21-activated kinases is controlled by the disordered variable region and polybasic sequences

p21-activated kinases (PAKs) are serine/threonine kinase effectors of the small GTPases Rac and Cdc42 and major participants in cell adhesion, motility, and survival. Type II PAKs (PAK4, -5, and -6) are recruited to cell-cell boundaries, where they regulate adhesion dynamics and colony escape. In contrast, the type I PAK, PAK1, does not localize to cell-cell contacts. We have now found that the other type I PAKs (PAK2 and PAK3) also fail to target to cell-cell junctions. PAKs contain extensive similarities in sequence and domain organization; therefore, focusing on PAK1 and PAK6, we used chimeras and truncation mutants to investigate their differences in localization. We observed that a weakly conserved sequence region (the variable region), located between the Cdc42-binding CRIB domain and the kinase domain, inhibits PAK1 targeting to cell-cell junctions. Accordingly, substitution of the PAK1 variable region with that from PAK6 or removal of this region of PAK1 resulted in its localization to cell-cell contacts. We further show that Cdc42 binding is required, but not sufficient, to direct PAKs to cell-cell contacts and that an N-terminal polybasic sequence is necessary for PAK1 recruitment to cell-cell contacts, but only if the variable region-mediated inhibition is released. We propose that all PAKs contain cell-cell boundary-targeting motifs but that the variable region prevents type I PAK accumulation at junctions. This highlights the importance of this poorly conserved, largely disordered region in PAK regulation and raises the possibility that variable region inhibition may be released by cellular signals.

Cdc42 defines apical identity and regulates epithelial morphogenesis by promoting apical recruitment of Par6-aPKC and Crumbs.

ABSTRACTCdc42 regulates epithelial morphogenesis together with the Par complex (Baz/Par3-Par6-aPKC), Crumbs (Crb/CRB3) and Stardust (Sdt/PALS1). However, how these proteins work together and interact during epithelial morphogenesis is not well understood. To address this issue, we used the genetically amenable Drosophila pupal photoreceptor and follicular epithelium. We show that during epithelial morphogenesis active Cdc42 accumulates at the developing apical membrane and cell-cell contacts, independently of the Par complex and Crb. However, membrane localization of Baz, Par6-aPKC and Crb all depend on Cdc42. We find that although binding of Cdc42 to Par6 is not essential for the recruitment of Par6 and aPKC to the membrane, it is required for their apical localization and accumulation, which we find also depends on Par6 retention by Crb. In the pupal photoreceptor, membrane recruitment of Par6-aPKC also depends on Baz. Our work shows that Cdc42 is required for this recruitment and suggests that this factor promotes the handover of Par6-aPKC from Baz onto Crb. Altogether, we propose that Cdc42 drives morphogenesis by conferring apical identity, Par-complex assembly and apical accumulation of Crb.

Regulation of Cdc42 and its effectors in epithelial morphogenesis.

Cdc42 - a member of the small Rho GTPase family - regulates cell polarity across organisms from yeast to humans. It is an essential regulator of polarized morphogenesis in epithelial cells, through coordination of apical membrane morphogenesis, lumen formation and junction maturation. In parallel, work in yeast and Caenorhabditiselegans has provided important clues as to how this molecular switch can generate and regulate polarity through localized activation or inhibition, and cytoskeleton regulation. Recent studies have revealed how important and complex these regulations can be during epithelial morphogenesis. This complexity is mirrored by the fact that Cdc42 can exert its function through many effector proteins. In epithelial cells, these include atypical PKC (aPKC, also known as PKC-3), the P21-activated kinase (PAK) family, myotonic dystrophy-related Cdc42 binding kinase beta (MRCKβ, also known as CDC42BPB) and neural Wiskott-Aldrich syndrome protein (N-WASp, also known as WASL). Here, we review how the spatial regulation of Cdc42 promotes polarity and polarized morphogenesis of the plasma membrane, with a focus on the epithelial cell type.

Solution structures and biophysical analysis of full-length group A PAKs reveal they are monomeric and auto-inhibited in cis.

The group A p21-activated kinases (PAKs) exist in an auto-inhibited form until activated by GTPase binding and auto-phosphorylation. In the auto-inhibited form, a regulatory domain binds to the kinase domain (KD) blocking the binding of substrates, and CDC42 or Rac binding to the regulatory domain relieves this auto-inhibition allowing auto-phosphorylation on the KD activation loop. We have determined the crystal structure of the PAK3 catalytic domain and by small angle X-ray scattering, the solution-phase structures of full-length inactive PAK1 and PAK3. The structures reveal a compact but elongated molecular shape that demonstrates that, together with multiple independent biophysical measurements and in contrast with previous assumptions, group A PAKs are monomeric both before and after activation, consistent with an activation mechanism of cis-auto-inhibition and initial cis-auto-phosphorylation, followed by transient dimerisation to allow trans-auto-phosphorylation for full activation, yielding a monomeric active PAK protein.

The Cdc42 effectors Gic1 and Gic2 regulate polarized post-Golgi secretion

BackgroundCell polarity refers to spatial difference in morphology, structure, and function within different parts of a single cell, which plays important roles in a wide range of cellular processes. In eukaryotic cells, the small GTPase Cdc42 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) are critical components for cell polarity and required for polarized exocytosis and cell growth. Previous data showed that the GTPase-interacting components, Gic1 and Gic2, control cell polarity through its binding with Cdc42 and PtdIns(4,5)P2 in the plasma membrane in budding yeast. However, whether the Gic proteins regulate polarized exocytosis is unknown.ResultsIn this study, we found that Gic2 co-immunoprecipitates with the exocyst complex, suggesting Gic proteins may be involved in exocytosis. Although we could not show the direct interaction between Gic2 and exocyst, we found gic1Δgic2Δ are synthetically sick with sec3ΔN. We demonstrated that Gic1 and Gic2 are required for polarized exocytosis in a yeast strain harboring the N-terminal domain deletion of Sec3, which is also known as an effector of Cdc42 GTPase. Gic proteins are required for polarized localization of exocyst, growth, and efficient secretion in sec3∆N mutant. In addition, we found that the N-terminal domain of both Gic2 and Sec3 share the similar binding sites of Cdc42. Surprisingly, not all the Sec3/Gic binding deficient cdc42 mutants displayed defects of growth and secretion, indicating that disruption of Cdc42 binding with Gic proteins and Sec3 does not necessarily show secretion defects in cdc42 mutants.ConclusionsWe conclude that Gic1/2 and Sec3 act in parallel to regulate polarized post-Golgi secretion, but this regulation is not solely controlled by their upstream factor Cdc42. Considering that N-terminal domain of Gic2 and Sec3 can bind to both Cdc42 and PtdIns(4,5)P2, the regulation of Gic protein and Sec3 on polarized secretion may also be controlled by PtdIns(4,5)P2. Further experiments need to be performed to test this hypothesis. Our findings provide important clues for understanding the molecular mechanism of cell polarity establishment in eukaryotic cells.