Effector PAK1 Research Articles

Pathophysiological significance of the p.E31G variant in RAC1 responsible for a neurodevelopmental disorder with microcephaly

RAC1 encodes a Rho family small GTPase that regulates actin cytoskeletal reorganization and intracellular signaling pathways. Pathogenic RAC1 variants lead to a neurodevelopmental disorder with diverse phenotypic manifestations, including abnormalities in brain size and facial dysmorphism. However, the underlying pathophysiological mechanisms have yet to be elucidated. Here, we present the case of a school-aged male who exhibited global developmental delay, intellectual disability, and acquired microcephaly. Through whole exome sequencing, we identified a novel de novo variant in RAC1, (NM_006908.5): c.92 A > G,p.(E31G). We then examined the pathophysiological significance of the p.E31G variant by focusing on brain development. Biochemical analyses revealed that the recombinant RAC1-E31G had no discernible impact on the intrinsic GDP/GTP exchange activity. However, it exhibited a slight inhibitory effect on GTP hydrolysis. Conversely, it demonstrated a typical response to both a guanine-nucleotide exchange factor and a GTPase-activating protein. In transient expression analyses using COS7 cells, RAC1-E31G exhibited minimal interaction with the downstream effector PAK1, even in its GTP-bound state. Additionally, overexpression of RAC1-E31G was observed to exert a weak inhibitory effect on the differentiation of primary cultured hippocampal neurons. Moreover, in vivo studies employing in utero electroporation revealed that acute expression of RAC1-E31G resulted in impairments in axonal elongation and dendritic arborization in the young adult stage. These findings suggest that the p.E31G variant functions as a dominant-negative version in the PAK1-mediated signaling pathway and is responsible for the clinical features observed in the patient under investigation, namely microcephaly and intellectual disability.

The Rac1-PAK1-Arp2/3 signaling axis regulates CHIKV nsP1-induced filopodia and optimal viral genome replication.

Alphavirus infection induces dramatic remodeling of host cellular membranes, producing filopodia-like and intercellular extensions. The formation of filopodia-like extensions has been primarily assigned to the replication protein nsP1, which binds and reshapes the host plasma membrane when expressed alone. While reported decades ago, the molecular mechanisms behind nsP1 membrane deformation remain unknown. Using mammalian epithelial cells and Chikungunya virus (CHIKV) as models, we characterized nsP1-induced membrane deformations as highly dynamic actin-rich lamellipodia and filopodia-like extensions. Through pharmacological inhibition and genetic invalidation, we identified the critical contribution of the Rac1 GTPase and its downstream effectors PAK1 and the actin nucleator Arp2 in nsP1-induced membrane deformation. An intact Rac1-PAK1-Arp2 signaling axis was also required for optimal CHIKV genome replication. Therefore, our results designate the Rac1-PAK1-Arp2 pathway as an essential signaling node for CHIKV infection and establish a parallel requirement for host factors involved in nsP1-induced plasma membrane reshaping and assembly of a functional replication complex.IMPORTANCEThe alphavirus nsP1 protein dramatically remodels host cellular membranes, resulting in the formation of filopodia-like extensions. Although described decades ago, the molecular mechanisms controlling these membrane deformations and their functional importance remain elusive. Our study provides mechanistic insight, uncovering the critical role of the Rac1 GTPase, along with its downstream effectors PAK1 and the actin nucleator Arp2, in the nsP1-associated phenotype. Furthermore, we demonstrate that the Rac1-PAK1-Arp2 pathway is essential for optimal CHIKV genome replication. Our findings establish a parallel in the cellular mechanisms governing nsP1-induced plasma membrane reshaping and the production of a functional replication complex in infected cells.

A ubiquitin-based effector-to-inhibitor switch coordinates early brain, craniofacial, and skin development

The molecular mechanisms that coordinate patterning of the embryonic ectoderm into spatially distinct lineages to form the nervous system, epidermis, and neural crest-derived craniofacial structures are unclear. Here, biochemical disease-variant profiling reveals a posttranslational pathway that drives early ectodermal differentiation in the vertebrate head. The anteriorly expressed ubiquitin ligase CRL3-KLHL4 restricts signaling of the ubiquitous cytoskeletal regulator CDC42. This regulation relies on the CDC42-activating complex GIT1-βPIX, which CRL3-KLHL4 exploits as a substrate-specific co-adaptor to recognize and monoubiquitylate PAK1. Surprisingly, we find that ubiquitylation converts the canonical CDC42 effector PAK1 into a CDC42 inhibitor. Loss of CRL3-KLHL4 or a disease-associated KLHL4 variant reduce PAK1 ubiquitylation causing overactivation of CDC42 signaling and defective ectodermal patterning and neurulation. Thus, tissue-specific restriction of CDC42 signaling by a ubiquitin-based effector-to-inhibitor is essential for early face, brain, and skin formation, revealing how cell-fate and morphometric changes are coordinated to ensure faithful organ development.

Modulation of Rac1/PAK1/connexin43-mediated ATP release from astrocytes contributes to retinal ganglion cell survival in experimental glaucoma.

Connexin43 (Cx43) is a major gap junction protein in glial cells. Mutations have been found in the gap-junction alpha 1 gene encoding Cx43 in glaucomatous human retinas, suggestive of the involvement of Cx43 in the pathogenesis of glaucoma. However, how Cx43 is involved in glaucoma is still unknown. We showed that increased intraocular pressure in a glaucoma mouse model of chronic ocular hypertension (COH) downregulated Cx43, which was mainly expressed in retinal astrocytes. Astrocytes in the optic nerve head where they gather and wrap the axons (optic nerve) of retinal ganglion cells (RGCs) were activated earlier than neurons in COH retinas and the alterations in astrocytes plasticity in the optic nerve caused a reduction in Cx43 expression. A time course showed that reductions of Cx43 expression were correlated with the activation of Rac1, a member of the Rho family. Co-immunoprecipitation assays showed that active Rac1, or the downstream signaling effector PAK1, negatively regulated Cx43 expression, Cx43 hemichannel opening and astrocyte activation. Pharmacological inhibition of Rac1 stimulated Cx43 hemichannel opening and ATP release, and astrocytes were identified to be one of the main sources of ATP. Furthermore, conditional knockout of Rac1 in astrocytes enhanced Cx43 expression and ATP release, and promoted RGC survival by upregulating the adenosine A3 receptor in RGCs. Our studyprovides new insight into the relationship between Cx43 and glaucoma, and suggests that regulating the interaction between astrocytes and RGCs via the Rac1/PAK1/Cx43/ATP pathway may be used as part of a therapeutic strategy for managing glaucoma.

MICAL1 activation by PAK1 mediates actin filament disassembly.

The MICAL1 monooxygenase is an important regulator of filamentous actin (F-actin) structures. Although MICAL1 has been shown to be regulated via protein-protein interactions at the autoinhibitory carboxyl terminus, a link between actin-regulatory RHO GTPase signaling pathways and MICAL1 has not been established. We show that the CDC42 GTPase effector PAK1 associates with and phosphorylates MICAL1 on two serine residues, leading to accelerated F-actin disassembly. PAK1 binds to the amino-terminal catalytic monooxygenase and calponin homology domains, distinct from the autoinhibitory carboxyl terminus. Extracellular ligand stimulation leads to PAK-dependent phosphorylation, linking external signals to MICAL1 phosphorylation. Mass spectrometry indicates that MICAL1 co-expression with CDC42 and PAK1 increases MICAL1 association with hundreds of proteins, including the previously described MICAL1-interacting proteins RAB10 and RAB7A. These results provide insights into a redox-mediated pathway linking extracellular signals to cytoskeleton regulation via a RHO GTPase and indicate a means of communication between RHO and RAB GTPases.

Abstract 1864: Baicalein attenuates endometrial cancer growth by suppressing the ARF6

Abstract Using mass spectrometry-based proteomics, we have previously shown enhanced expression of the Adenosine diphosphate-Ribosylation Factor-6 (ARF6) in endometrial cancer cell lines compared to immortalized EM-E6/E7-TERT endometrial epithelial cells. ARFs belong to the RAS superfamily of small GTPases. ARF family proteins are activated by Guanine nucleotide Exchange Factors (GEFs) and inactivated by GTPase-Activating Proteins (GAPs). When active, they bind effectors, which mediate downstream functions. ARF6 localized on the plasma membrane plays a role in actin cytoskeleton dynamics and endocytic recycling. Dysregulation of expression and/or activity of ARF6 has been associated with enhanced cell migration, invasion, and proliferation in several types of cancer. Knockdown of ARF6 significantly inhibited the invasiveness of the cancer cells. There is a critical need for novel promising anticancer agents with lesser side effects and higher efficacy. Baicalein, extracted from the roots of the Chinese herb Huang Qin (Scutellaria baicalensis), showed a significant anticancer impact on many human cancers. However, the specific molecular mechanisms are still nebulous. Endometrial cancer cell lines were exposed to different concentrations of baicalein for 24 to 120 h. The effect on cell proliferation and invasion was determined utilizing MTS and Boyden chamber assays, respectively. The baicalein induced a dose and time-dependent reduction in cell viability. The invasiveness of cells was attenuated by baicalein. Furthermore, the anticancer effects were mediated by decreased ARF6, Rac1, and PAK1 expression in baicalein-treated cancer cells. In summary, the data reveal that suppression of ARF6 could reduce the invasive potential of endometrial cancer by modulating the major downstream effectors Rac1 and PAK1. Citation Format: Latoya Mcglorthan, Viqar Syed. Baicalein attenuates endometrial cancer growth by suppressing the ARF6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1864.

Alchemical Free Energy Calculations to Investigate Protein-Protein Interactions: the Case of the CDC42/PAK1 Complex.

Here, we show that alchemical free energy calculations can quantitatively compute the effect of mutations at the protein–protein interface. As a test case, we have used the protein complex formed by the small Rho-GTPase CDC42 and its downstream effector PAK1, a serine/threonine kinase. Notably, the CDC42/PAK1 complex offers a wealth of structural, mutagenesis, and binding affinity data because of its central role in cellular signaling and cancer progression. In this context, we have considered 16 mutations in the CDC42/PAK1 complex and obtained excellent agreement between computed and experimental data on binding affinity. Importantly, we also show that a careful analysis of the side-chain conformations in the mutated amino acids can considerably improve the computed estimates, solving issues related to sampling limitations. Overall, this study demonstrates that alchemical free energy calculations can conveniently be integrated into the design of experimental mutagenesis studies.

Rac1-PAK1 regulation of Rab11 cycling promotes junction destabilization

Rac1 GTPase is hyperactivated in tumors and contributes to malignancy. Rac1 disruption of junctions requires its effector PAK1, but the precise mechanisms are unknown. Here, we show that E-cadherin is internalized via micropinocytosis in a PAK1-dependent manner without catenin dissociation and degradation. In addition to internalization, PAK1 regulates E-cadherin transport by fine-tuning Rab small GTPase function. PAK1 phosphorylates a core Rab regulator, RabGDIβ, but not RabGDIα. Phosphorylated RabGDIβ preferentially associates with Rab5 and Rab11, which is predicted to promote Rab retrieval from membranes. Consistent with this hypothesis, Rab11 is activated by Rac1, and inhibition of Rab11 function partially rescues E-cadherin destabilization. Thus, Rac1 activation reduces surface cadherin levels as a net result of higher bulk flow of membrane uptake that counteracts Rab11-dependent E-cadherin delivery to junctions (recycling and/or exocytosis). This unique small GTPase crosstalk has an impact on Rac1 and PAK1 regulation of membrane remodeling during epithelial dedifferentiation, adhesion, and motility.

Abstract 5324: Structure-based design of CDC42/RHOJ effector inhibitors for the treatment of cancer

Abstract The CDC42 family of GTPases (RHOJ, RHOQ, CDC42) control both the ability of tumor cells to invade surrounding tissues and the ability of endothelial cells to vascularize tumors. While recent studies have developed small molecules that target RAS GTPases, little progress has been made in targeting the related CDC42 family of GTPases for cancer treatment. Here, we use computer-aided drug design to identify a novel class of inhibitors that act on an allosteric pocket in the active form of the CDC42 GTPase, RHOJ. These allosteric inhibitors prevent RHOJ and CDC42 from binding to their downstream effector PAK, while having no effect on the interactions between the closely related GTPase RAC1 and PAK or RAS and its downstream effector RAF. Our lead compound ARN22089 has a druglike profile and can block both tumor growth and tumor angiogenesis in a three-dimensional vascularized microtumor (VMT) model, indicating that ARN22089 blocks RHOJ/CDC42 signaling in both the tumor cell and the tumor endothelium. Short term treatment of nascent melanoma tumors with ARN22089 halted the growth of BRAF mutant autochthonous mouse melanoma tumors, slowed the growth of melanoma patient-derived xenografts, and induced tumor necrosis in PDX models. In summary, we describe a multidisciplinary structure-based drug discovery platform that can identify new RHO family allosteric inhibitors and use this system to identify RHOJ inhibitors that block tumor growth in vivo. Citation Format: Jessica L. Flesher, Sohail Jahid, Jose A. Ortega, Giuseppina La Sala, Nicoletta Brindani, Jose M. Arencibia, Jacopo Manigrasso, Stephanie Hachey, Chi-Fen Chen, Chris Hughes, Marco De Vivo, Anand K. Ganesan. Structure-based design of CDC42/RHOJ effector inhibitors for the treatment of cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5324.

Endophilin-A2 dependent VEGFR2 endocytosis promotes sprouting angiogenesis

Endothelial cell migration, proliferation and survival are triggered by VEGF-A activation of VEGFR2. However, how these cell behaviors are regulated individually is still unknown. Here we identify Endophilin-A2 (ENDOA2), a BAR-domain protein that orchestrates CLATHRIN-independent internalization, as a critical mediator of endothelial cell migration and sprouting angiogenesis. We show that EndoA2 knockout mice exhibit postnatal angiogenesis defects and impaired front-rear polarization of sprouting tip cells. ENDOA2 deficiency reduces VEGFR2 internalization and inhibits downstream activation of the signaling effector PAK but not ERK, thereby affecting front-rear polarity and migration but not proliferation or survival. Mechanistically, VEGFR2 is directed towards ENDOA2-mediated endocytosis by the SLIT2-ROBO pathway via SLIT-ROBO-GAP1 bridging of ENDOA2 and ROBO1. Blocking ENDOA2-mediated endothelial cell migration attenuates pathological angiogenesis in oxygen-induced retinopathy models. This work identifies a specific endocytic pathway controlling a subset of VEGFR2 mediated responses that could be targeted to prevent excessive sprouting angiogenesis in pathological conditions.

Balanced Rac1 activity controls formation and maintenance of neuromuscular acetylcholine receptor clusters.

Rac1, an important Rho GTPase that regulates the actin cytoskeleton, has long been suggested to participate in acetylcholine receptor (AChR) clustering at the postsynaptic neuromuscular junction. However, how Rac1 is regulated and how it influences AChR clusters have remained unexplored. This study shows that breaking the balance of Rac1 regulation, by either increasing or decreasing its activity, led to impaired formation and maintenance of AChR clusters. By manipulating Rac1 activity at different stages of AChR clustering in cultured myotubes, we show that Rac1 activation was required for the initial formation of AChR clusters, but its persistent activation led to AChR destabilization, and uncontrolled hyperactivation of Rac1 even caused excessive myotube fusion. Both AChR dispersal and myotube fusion induced by Rac1 were dependent on its downstream effector Pak1. Two Rac1 GAPs and six Rac1 GEFs were screened and found to be important for normal AChR clustering. This study reveals that, although general Rac1 activity remains at low levels during terminal differentiation of myotubes and AChR cluster maintenance, tightly regulated Rac1 activity controls normal AChR clustering.

Migracja – regulacja zjawiska przez wybrane szlaki sygnałowe

Ruch i migracja są jedną z głównych funkcji życiowych komórek. W odpowiedzi na różne bodźce, dynamiczny cytoszkielet aktynowy generuje siłę umożliwiającą komórce przemieszczanie się w trójwymiarowej sieci zewnątrzkomórkowej macierzy czy po płaskim podłożu. Wydłużanie filamentów aktynowych na ich kolczastych końcach wypycha błonę komórkową w kierunku migracji, formując strefę frontalną zwaną lamellipodium. Skurcz włókien naprężeniowych umożliwia oderwanie tylnej części komórki i przesunięcie jej do przodu. W odpowiedzi na bodźce ze środowiska, receptory komórki inicjują wiele szlaków sygnałowych powodujących reorganizację mikrofilamentów aktynowych oraz skurcz układu akto-miozynowego. Głównymi regulatorami tych procesów są białka z rodziny Rho, fosfolipidy PIP2 oraz jony wapnia. Receptory nukleotydowe P2Y2 w połączeniu z białkami G regulują poziom fosfatydyloinozytolu-4,5-bisfosforanu (PIP2), który moduluje funkcje białek wiążących aktynę i aktywuje białka Rac1 oraz RhoA. Szlak sygnałowy RhoA/ROCK odgrywa ważną rolę w generowaniu skurczu włókien naprężeniowych. Z kolei białko Rac1 poprzez swój efektor kinazę PAK1 reguluje procesy formujące lamellipodium oraz wysuwanie strefy wiodącej podczas migracji.

α5β1 integrin trafficking and Rac activation are regulated by APPL1 in a Rab5-dependent manner to inhibit cell migration.

Cell migration is a tightly coordinated process that requires the spatiotemporal regulation of many molecular components. Because adaptor proteins can serve as integrators of cellular events, they are being increasingly studied as regulators of cell migration. The adaptor protein containing a pleckstrin-homology (PH) domain, phosphotyrosine binding (PTB) domain, and leucine zipper motif 1 (APPL1) is a 709 amino acid endosomal protein that plays a role in cell proliferation and survival as well as endosomal trafficking and signaling. However, its function in regulating cell migration is poorly understood. Here, we show that APPL1 hinders cell migration by modulating both trafficking and signaling events controlled by Rab5 in cancer cells. APPL1 decreases internalization and increases recycling of α5β1 integrin, leading to higher levels of α5β1 integrin at the cell surface that hinder adhesion dynamics. Furthermore, APPL1 decreases the activity of the GTPase Rac and its effector PAK, which in turn regulate cell migration. Thus, we demonstrate a novel role for the interaction between APPL1 and Rab5 in governing crosstalk between signaling and trafficking pathways on endosomes to affect cancer cell migration.This article has an associated First Person interview with the first author of the paper.

Infectious pancreatic necrosis virus enters CHSE-214 cells via macropinocytosis

Infectious pancreatic necrosis virus (IPNV) is a non-enveloped virus belonging to the Birnaviridae family. IPNV produces an acute disease in salmon fingerlings, with high mortality rates and persistent infection in survivors. Although there are reports of IPNV binding to various cells, the viral receptor and entry pathways remain unknown. The aim of this study was to determine the endocytic pathway that allows for IPNV entry. We observed that IPNV stimulated fluid uptake and virus particles co-localysed with the uptake marker dextran in intracellular compartments, suggesting a role for macropinocytosis in viral entry. Consistent with this idea, viral infection was significantly reduced when the Na+/H+ exchanger NHE1 was inhibited with 5-(N-Ethyl-N-isopropyl) amiloride (EIPA). Neither chlorpromazine nor filipin complex I affected IPNV infection. To examine the role of macropinocytosis regulators, additional inhibitors were tested. Inhibitors of the EGFR pathway and the effectors Pak1, Rac1 and PKC reduced viral infection. Together, our results indicate that IPNV is mainly internalized into CHSE-214 cells by macropinocytosis.

Discovery and characterization of small molecule Rac1 inhibitors

Aberrant activation of Rho GTPase Rac1 has been observed in various tumor types, including pancreatic cancer. Rac1 activates multiple signaling pathways that lead to uncontrolled proliferation, invasion and metastasis. Thus, inhibition of Rac1 activity is a viable therapeutic strategy for proliferative disorders such as cancer. Here we identified small molecule inhibitors that target the nucleotide-binding site of Rac1 through in silico screening. Follow up in vitro studies demonstrated that two compounds blocked active Rac1 from binding to its effector PAK1. Fluorescence polarization studies indicate that these compounds target the nucleotide-binding site of Rac1. In cells, both compounds blocked Rac1 binding to its effector PAK1 following EGF-induced Rac1 activation in a dose-dependent manner, while showing no inhibition of the closely related Cdc42 and RhoA activity. Furthermore, functional studies indicate that both compounds reduced cell proliferation and migration in a dose-dependent manner in multiple pancreatic cancer cell lines. Additionally, the two compounds suppressed the clonogenic survival of pancreatic cancer cells, while they had no effect on the survival of normal pancreatic ductal cells. These compounds do not share the core structure of the known Rac1 inhibitors and could serve as additional lead compounds to target pancreatic cancers with high Rac1 activity.

Role of End Binding Protein-1 in endothelial permeability response to barrier-disruptive and barrier-enhancing agonists

Rapid changes in microtubule (MT) polymerization dynamics affect regional activity of small GTPases RhoA and Rac1, which play a key role in the regulation of actin cytoskeleton and endothelial cell (EC) permeability. This study tested the role of End Binding Protein-1 (EB1) in the mechanisms of increased and decreased EC permeability caused by thrombin and hepatocyte growth factor (HGF) and mediated by RhoA and Rac1 GTPases, respectively. Stimulation of human lung EC with thrombin inhibited peripheral MT growth, which was monitored by morphological and biochemical evaluation of peripheral MT and the levels of stabilized MT. In contrast, stimulation of EC with HGF promoted peripheral MT growth and protrusion of EB1-positive MT plus ends to the EC peripheral submembrane area. EB1 knockdown by small interfering RNA did not affect partial MT depolymerization, activation of Rho signaling, and permeability response to thrombin, but suppressed the HGF-induced endothelial barrier enhancement. EB1 knockdown suppressed HGF-induced activation of Rac1 and Rac1 cytoskeletal effectors cortactin and PAK1, impaired HGF-induced assembly of cortical cytoskeleton regulatory complex (WAVE-p21Arc-IQGAP1), and blocked HGF-induced enhancement of peripheral actin cytoskeleton and VE-cadherin-positive adherens junctions. Altogether, these data demonstrate a role for EB1 in coordination of MT-dependent barrier enhancement response to HGF, but show no involvement of EB1 in acute increase of EC permeability caused by the barrier disruptive agonist. The results suggest that increased peripheral EB1 distribution is a critical component of the Rac1-mediated pathway and peripheral cytoskeletal remodeling essential for agonist-induced EC barrier enhancement.

The chloride intracellular channel 5A stimulates podocyte Rac1, protecting against hypertension-induced glomerular injury

Glomerular capillary hypertension elicits podocyte remodeling and is a risk factor for the progression of glomerular disease. Ezrin, which links podocalyxin to actin in podocytes, is activated through the chloride intracellular channel 5A (CLIC5A)-dependent phosphatidylinositol 4,5 bisphosphate (PI[4,5]P2) accumulation. Because Rac1 is involved in podocyte actin remodeling and can promote PI[4,5]P2 production we determined whether CLIC5A-dependent PI[4,5]P2 generation and ezrin activation are mediated by Rac1. In COS7 cells, CLIC5A expression stimulated Rac1 but not Cdc42 or Rho activity. CLIC5A alsostimulated phosphorylation of the Rac1 effector Pak1 in COS7 cells and in cultured mouse podocytes. CLIC5A-induced PI[4,5]P2 accumulation and Pak1 and ezrin phosphorylation were all Rac1 dependent. In DOCA/Salt hypertension, phosphorylated Pak increased in podocytes of wild-type, but not CLIC5-deficient mice. In DOCA/salt hypertensive mice lacking CLIC5, glomerular capillary microaneurysms were more frequent and albuminuria wasgreater than in wild-type mice. Thus, augmented hypertension-induced glomerular capillary injury in mice lacking CLIC5 results from abrogation of Rac1-dependent Pak and ezrin activation, perhaps reducing the tensile strength of the podocyte actin cytoskeleton.

Neuronal filopodium formation induced by the membrane glycoprotein M6a (Gpm6a) is facilitated by coronin‐1a, Rac1, and p21‐activated kinase 1 (Pak1)

Stress-responsive neuronal membrane glycoprotein M6a (Gpm6a) functions in neurite extension, filopodium and spine formation and synaptogenesis. The mechanisms of Gpm6a action in these processes are incompletely understood. Previously, we identified the actin regulator coronin-1a (Coro1a) as a putative Gpm6a interacting partner. Here, we used co-immunoprecipitation assays with the anti-Coro1a antibody to show that Coro1a associates with Gpm6a in rat hippocampal neurons. By immunofluorescence microscopy, we demonstrated that in hippocampal neurons Coro1a localizes in F-actin-enriched regions and some of Coro1a spots co-localize with Gpm6a labeling. Notably, the over-expression of a dominant-negative form of Coro1a as well as its down-regulation by siRNA interfered with Gpm6a-induced filopodium formation. Coro1a is known to regulate the plasma membrane translocation and activation of small GTPase Rac1. We show that Coro1a co-immunoprecipitates with Rac1 together with Gpm6a. Pharmacological inhibition of Rac1 resulted in a significant decrease in filopodium formation by Gpm6a. The same was observed upon the co-expression of Gpm6a with the inactive GDP-bound form of Rac1. In this case, the elevated membrane recruitment of GDP-bound Rac1 was detected as well. Moreover, the kinase activity of the p21-activated kinase 1 (Pak1), a main downstream effector of Rac1 that acts downstream of Coro1a, was required for Gpm6a-induced filopodium formation. Taken together, our results provide evidence that a signaling pathway including Coro1a, Rac1, and Pak1 facilitates Gpm6a-induced filopodium formation. Formation of filopodia by membrane glycoprotein M6a (Gpm6a) requires actin regulator coronin-1a (Coro1a), known to regulate plasma membrane localization and activation of Rac1 and its downstream effector Pak1. Coro1a associates with Gpm6a. Blockage of Coro1a, Rac1, or Pak1 interferes with Gpm6a-induced filopodium formation. Moreover, Gpm6a facilitates Rac1 membrane recruitment. Altogether, a mechanistic insight into the process of Gpm6a-induced neuronal filopodium formation is provided.

Specification of Dendritogenesis Site in Drosophila aCC Motoneuron by Membrane Enrichment of Pak1 through Dscam1

Precise positioning of dendritic branches is a critical step in the establishment of neuronal circuitry. However, there is limited knowledge on how environmental cues translate into dendrite initiation or branching at a specific position. Here, through a combination of mutation, RNAi, and imaging experiments, we found that a Dscam-Dock-Pak1 hierarchical interaction defines the stereotypical dendrite growth site in the Drosophila aCC motoneuron. This interaction localizes the Cdc42 effector Pak1 to the plasma membrane at the dendrite initiation site before the activation of Cdc42. Ectopic expression of membrane-anchored Pak1 overrides this spatial specification of dendritogenesis, confirming its function in guiding Cdc42 signaling. We further discovered that Dscam1 localization in aCC occurs through an inter-neuronal contact that involves Dscam1 in the partner MP1 neuron. These findings elucidate a mechanism by which Dscam1 controls neuronal morphogenesis through spatial regulation of Cdc42 signaling and, subsequently, cytoskeletal remodeling.

Regulation of Stat5 by FAK and PAK1 in Oncogenic FLT3- and KIT-Driven Leukemogenesis

Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs), and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription, is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK) whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis.