FAK dimerization controls its kinase‐dependent functions at focal adhesions

Focal adhesion kinase (FAK) controls adhesion‐dependent cell motility, survival, and proliferation. FAK has kinase‐dependent and kinase‐independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x‐ray crystallography, small angle x‐ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase‐dependent functions—autophosphorylation of tyrosine‐397—requires site‐specific dimerization of FAK. The dimers form via the association of the N‐terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C‐terminal FAT domain. FAT binds to a basic motif on FERM that regulates co‐activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site‐specific function. The dimer interfaces we describe are promising targets for blocking FAK activation.     

Alveolar type II cells escape stress failure caused by tonic stretch through transient focal adhesion disassembly

Mechanical ventilation-induced excessive stretch of alveoli is reported to induce cellular stress failure and subsequent lung injury, and is therefore an injurious factor to the lung. Avoiding cellular stress failure is crucial to ventilator-induced lung injury (VILI) treatment. In the present study, primary rat alveolar type II (ATII) cells were isolated to evaluate their viability and the mechanism of their survival under tonic stretch. By the annexin V/ PI staining and flow cytometry assay, we demonstrated that tonic stretch-induced cell death is an immediate injury of mechanical stress. In addition, immunofluorescence and immunoblots assay showed that the cells experienced an expansion-contraction-reexpansion process, accompanied by partial focal adhesion (FA) disassembly during contraction. Manipulation of integrin adherent affinity by altering bivalent cation levels in the culture medium and applying an integrin neutralizing antibody showed that facilitated adhesion affinity promoted cell death under tonic stretch, while lower level of adhesion protected the cells from stretch-induced stress failure. Finally, a simplified numerical model was established to reveal that adequate disassembly of FAs reduced the forces transmitting throughout the cell. Taken together, these results indicate that ATII cells escape stress failure caused by tonic stretch via active cell morphological remodeling, during which cells transiently disassemble FAs to unload mechanical forces.     

High tidal volume mechanical ventilation with hyperoxia alters alveolar type II cell adhesion

Patients with acute respiratory distress syndrome undergoing mechanical ventilation may be exposed to both high levels of stretch and high levels of oxygen. We hypothesized that the combination of high stretch and hyperoxia promotes loss of epithelial adhesion and impairs epithelial repair mechanisms necessary for restoration of barrier function. We utilized a model of high tidal volume mechanical ventilation (25 ml/kg) with hyperoxia (50% O(2)) in rats to investigate alveolar type II (AT2) cell adhesion and focal adhesion signaling. AT2 cells isolated from rats exposed to hyperoxia and high tidal volume mechanical ventilation (MVHO) exhibited significantly decreased cell adhesion and reduction in phosphotyrosyl levels of focal adhesion kinase (FAK) and paxillin compared with control rats, rats exposed to hyperoxia without ventilation (HO), or rats ventilated with normoxia (MV). MV alone increased phosphorylation of p130(Cas). RhoA activation was increased by MV, HO, and the combination of MV and HO. Treatment of MVHO cells with keratinocyte growth factor (KGF) for 1 h upon isolation reduced RhoA activity and restored attachment to control levels. Attachment and migration of control AT2 cells was significantly decreased by constitutively active RhoA or a kinase inactive form of FAK (FRNK), whereas expression of dominant negative RhoA in cells from MVHO-treated rats restored cell adhesion. Mechanical ventilation with hyperoxia promotes changes in focal adhesion proteins and RhoA in AT2 cells that may be deleterious for cell adhesion and migration.     

Focal adhesion kinase localizes to sites of cell‐to‐cell contact in vivo and increases apically in rat uterine luminal epithelium and the blastocyst at the time of implantation

Focal adhesions play an important role in promoting embryo invasion; in particular, focal adhesions disassemble at the time of implantation in the rat, facilitating the detachment of the uterine luminal epithelium to allow the embryo to invade the endometrium. This study investigated focal adhesion protein, focal adhesion kinase (FAK) in the rat uterine luminal, and glandular epithelial cells to understand the dynamics of focal adhesions during early pregnancy. FAK undergoes extensive distributional change during early pregnancy, and surprisingly, FAK was not localized at the site of focal adhesions, instead being localized to the site of cell‐to‐cell contact and colocalizing with ZO‐1 on day 1 of pregnancy. At the time of implantation, FAK increases in the apical region of the uterine luminal epithelial cells which was regulated by progesterone. Using an in vitro co‐culture model of rat blastocysts attached to Ishikawa cells, FAK was present apically both in the rat blastocyst and the Ishikawa cells, suggesting a role in attachment andin mediating signal transduction between these two genetically different cell types. J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

Hyperoxia-induced apoptosis and Fas/FasL expression in lung epithelial cells

Alveolar epithelial apoptosis is an important feature of hyperoxia-induced lung injury in vivo and has been described in the early stages of bronchopulmonary dysplasia (chronic lung disease of preterm newborn). Molecular regulation of hyperoxia-induced alveolar epithelial cell death remains incompletely understood. In view of functional involvement of Fas/FasL system in physiological postcanalicular type II cell apoptosis, we speculated this system may also be a critical regulator of hyperoxia-induced apoptosis. The aim of this study was to investigate the effects of hyperoxia on apoptosis and apoptotic gene expression in alveolar epithelial cells. Apoptosis was studied by TUNEL, electron microscopy, DNA size analysis, and caspase assays. Fas/FasL expression was determined by Western blot analysis and RPA. We determined that in MLE-12 cells exposed to hyperoxia, caspase-mediated apoptosis was the first morphologically and biochemically recognizable mode of cell death, followed by necrosis of residual adherent cells. The apoptotic stage was associated with a threefold upregulation of Fas mRNA and protein expression and increased susceptibility to direct Fas receptor activation, concomitant with a threefold increase of FasL protein levels. Fas gene silencing by siRNAs significantly reduced hyperoxia-induced apoptosis. In murine fetal type II cells, hyperoxia similarly induced markedly increased Fas/FasL protein expression, confirming validity of results obtained in transformed MLE-12 cells. Our findings implicate the Fas/FasL system as an important regulator of hyperoxia-induced type II cell apoptosis. Elucidation of regulation of hyperoxia-induced lung apoptosis may lead to alternative therapeutic strategies for perinatal or adult pulmonary diseases characterized by dysregulated type II cell apoptosis.     

Activation of canonical wnt pathway promotes differentiation of mouse bone marrow‐derived MSCs into type II alveolar epithelial cells,confers resistance to oxidative stress,and promotes their migration to injured lung tissue in vitro

The differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells in vivo and in vitro, is critical for reepithelization and recovery in acute lung injury (ALI), but the mechanisms responsible for differentiation are unclear. In the present study, we investigated the role of the canonical wnt pathway in the differentiation of mouse bone marrow‐derived MSCs (mMSCs) into AT II cells. Using a modified co‐culture system with murine lung epithelial‐12 (MLE‐12) cells and small airway growth media (SAGM) to efficiently drive mMSCs differentiation, we found that GSK 3β and β‐catenin in the canonical wnt pathway were up‐regulated during differentiation. The levels of surfactant protein (SP) C, SPB, and SPD, the specific markers of AT II cells, correspondingly increased in mMSCs when Wnt3a or LiCl was added to the co‐culture system to activate wnt/β‐catenin signaling. The expression of these factors was depressed to some extent by inhibiting the pathway with the addition of DKK 1. The differentiation rate of mMSCs also depends on their abilities to accumulate and survive in inflammatory tissue. Our results suggested that the activation of wnt/β‐catenin signaling promoted mMSCs migration towards ALI mouse‐derived lung tissue in a Transwell assay, and ameliorated the cell death and the reduction of Bcl‐2/Bax induced by H₂O₂, which simultaneously caused reduced GSK 3β and β‐catenin in mMSCs. These data supports a potential mechanism for the differentiation of mMSCs into AT II cells involving canonical wnt pathway activation, which may be significant to their application in ALI. J. Cell. Physiol. 228: 1270–1283, 2013. © 2012 Wiley Periodicals, Inc.     

Promotion of Skeletal Muscle Differentiation by K252a with Tyrosine Phosphorylation of Focal Adhesion: A Possible Involvement of Small GTPase Rho

K252a, a protein kinase inhibitor, acts as a neurotrophic factor in several neuronal cells. In this study we show that K252a enhanced the differentiation of C2C12 myoblasts as well as tyrosine phosphorylation of several focal adhesion-associated proteins including p130(Cas), focal adhesion kinase, and paxillin. The tyrosine phosphorylation of these proteins, reaching a maximum at 30 min after K252a treatment, closely correlated with the colocalization of these proteins in focal adhesion complexes and the coimmunoprecipitation of these proteins with p130(Cas). In addition, K252a stimulated longitudinal development of stress fiber-like structures and cell-matrix interaction in postmitotic myoblasts and eventually formation of well-developed myofibrils in multinucleated myotubes. Herbimycin A, a potent inhibitor of Src family kinases, and cytochalasin D, a selective disrupting-agent of actin filament, completely inhibited K252a-induced tyrosine phosphorylation as well as myoblast differentiation. Similar inhibitory effect was observed in the cells scrape loaded with a Rho inhibitor, C3 transferase, and the treatment of K252a induced a rapid translocation of Rho. These results are consistent with the model that Rho-dependent tyrosine phosphorylation of focal adhesion-associated proteins plays an important role in skeletal muscle differentiation.     

Dissecting focal adhesions in cells differentially expressing calreticulin: a microscopy study

Background information. Our previous studies have shown that calreticulin, a Ca²⁺‐binding chaperone located in the endoplasmic reticulum, affects cell—substratum adhesions via the induction of vinculin and N‐cadherin. Cells overexpressing calreticulin contain more vinculin than low expressers and make abundant contacts with the substratum. However, cells that express low levels of calreticulin exhibit a weak adhesive phenotype and make few, if any, focal adhesions. To date, the identity of the types of focal adhesions made by calreticulin overexpressing and low expressing cells has not been dissected. Results. The results of the present study show that calreticulin affects fibronectin matrix assembly in L fibroblast cell lines that differentially express the protein, and that these cells also differ profoundly in focal adhesion formation. Although the calreticulin overexpressing cells generate numerous interference‐reflection‐microscopy‐dark, vinculin‐ and paxillin‐containing classical focal contacts, as well as some fibrillar adhesions, the cells expressing low levels of calreticulin generate only a few weak focal adhesions. The fibronectin receptor was found to be clustered in calreticulin overexpressing cells, but diffusely distributed over the cell surface in low expressing cells. Plating L fibroblasts on fibronectin‐coated substrata induced extensive spreading in all cell lines tested. However, although calreticulin overexpressing cells were induced to form classical vinculin‐rich focal contacts, the low calreticulin expressing cells overcame their weak adhesive phenotype by induction of many tensin‐rich fibrillar adhesions, thus compensating for the low level of vinculin in these cells. Conclusions. We propose that calreticulin affects fibronectin production and, thereby, assembly, and it indirectly influences the formation and/or stability of focal contacts and fibrillar adhesions, both of which are instrumental in matrix assembly and remodelling.     

Spatial activation of ezrin by epidermal growth factor receptor and focal adhesion kinase co-ordinates epithelial cell migration

Epidermal growth factor receptor (EGFR) plays a critical role in the promotion of epithelial cell proliferation and migration. Previous studies have suggested a cooperative role between EGFR and integrin signalling pathways that enable efficient adhesion and migration but the mechanisms controlling this remain poorly defined. Here, we show that EGFR forms a complex with focal adhesion kinase in epithelial cells. Surprisingly, this complex enhances local Src activity at focal adhesions to promote phosphorylation of the cytoskeletal adaptor protein ezrin at Y478, leading to actomyosin contractility, suppression of focal adhesion dynamics and slower migration. We further demonstrate this regulation of Src is due to the suppression of PTP1B activity. Our data provide new insight into EGF-independent cooperation between EGFR and integrins and suggest transient interactions between these kinases at the leading edge of cells act to spatially control signalling to permit efficient motility.     

Parathyroid hormone-related protein response to hyperoxic lung injury

Parathyroid hormone-related protein (PTHrP) is a growth inhibitor for alveolar type II cells. Type II cell proliferation after lung injury from 85% oxygen is regulated, in part, by a fall in lung PTHrP. In this study, we investigated lung PTHrP after injury induced by >95% oxygen in rats and rabbits. In adult rats, lung PTHrP rose 10-fold over controls to 6,356 +/- 710 pg/ml (mean +/- SE) at 48 h of hyperoxia. Levels fell to 299 +/- 78 pg/ml, and staining for PTHrP mRNA was greatly reduced at 60 h (P < 0.05), the point of most severe injury and greatest pneumocyte proliferation. In adult rabbits, lung PTHrP peaked at 3,289 +/- 230 pg/ml after 64 h of hyperoxia with 24 h of normoxic recovery and then dropped to 1,629 +/- 153 pg/ml at 48 h of recovery (P < 0.05). Type II cell proliferation peaked shortly after the fall in PTHrP. In newborn rabbits, lavage PTHrP increased by 50% during the first 8 days of hyperoxia, whereas type II cell growth decreased. PTHrP declined at the LD(50), concurrent with increased type II cell division. In summary, lung PTHrP initially rises after injury with >95% hyperoxia and then falls near the peak of injury. Changes in PTHrP are temporally related to type II cell proliferation and may regulate repair of lung injury.     

Organization of focal adhesion plaques is disrupted by action of the HIV-1 protease

Focal adhesion plaques were severely affected in human embryonic fibroblasts permeabilized with digitonin and incubated in buffer containing the human immunodeficiency virus type 1 protease (HIV-1 PR). A mutant HIV-1 PR (3271 HIV-1 PR) had no effect on focal adhesion plaques. Similar effects were seen with cells microinjected with either HIV-1 PR or 3271 HIV-1 PR. Immunoblots of the human embryonic fibroblasts demonstrated that a number of focal adhesion plaque proteins were specifically cleaved by HIV-1 PR. These included fimbrin, focal adhesion plaque kinase (FAK), talin, and, to a lesser extent, filamin, spectrin and fibronectin. Proteins detected by antibodies to beta 4 integrin and alpha 3 integrin were also cleaved by the HIV-1 PR. Control experiments demonstrated that the effect and protein cleavages described are due to action of the HIV-1 PR and not to the action of endogenous host cell proteases.     

Hyperoxia alters the mechanical properties of alveolar epithelial cells

Patients with severe acute lung injury are frequently administered high concentrations of oxygen (>50%) during mechanical ventilation. Long-term exposure to high levels of oxygen can cause lung injury in the absence of mechanical ventilation, but the combination of the two accelerates and increases injury. Hyperoxia causes injury to cells through the generation of excessive reactive oxygen species. However, the precise mechanisms that lead to epithelial injury and the reasons for increased injury caused by mechanical ventilation are not well understood. We hypothesized that alveolar epithelial cells (AECs) may be more susceptible to injury caused by mechanical ventilation if hyperoxia alters the mechanical properties of the cells causing them to resist deformation. To test this hypothesis, we used atomic force microscopy in the indentation mode to measure the mechanical properties of cultured AECs. Exposure of AECs to hyperoxia for 24 to 48 h caused a significant increase in the elastic modulus (a measure of resistance to deformation) of both primary rat type II AECs and a cell line of mouse AECs (MLE-12). Hyperoxia also caused remodeling of both actin and microtubules. The increase in elastic modulus was blocked by treatment with cytochalasin D. Using finite element analysis, we showed that the increase in elastic modulus can lead to increased stress near the cell perimeter in the presence of stretch. We then demonstrated that cyclic stretch of hyperoxia-treated cells caused significant cell detachment. Our results suggest that exposure to hyperoxia causes structural remodeling of AECs that leads to decreased cell deformability.     

Calpain2 mediates Rab5-driven focal adhesion disassembly and cell migration

The early endosome protein Rab5 was recently shown to promote cell migration by enhancing focal adhesion disassembly through mechanisms that remain elusive. Focal adhesion disassembly is associated to proteolysis of talin, in a process that requires calpain2. Since calpain2 has been found at vesicles and endosomal compartments, we hypothesized that Rab5 stimulates calpain2 activity, leading to enhanced focal adhesion disassembly in migrating cells. We observed that calpain2 co-localizes with EEA1-positive early endosomes and co-immunoprecipitates with EEA1 and Rab5 in A549 lung carcinoma cells undergoing spreading, whereas Rab5 knock-down decreased the accumulation of calpain2 at early endosomal-enriched fractions. In addition, Rab5 silencing decreased calpain2 activity, as shown by cleavage of the fluorogenic substrate tBOC-LM-CMAC and the endogenous substrate talin. Accordingly, Rab5 promoted focal adhesion disassembly in a calpain2-dependent manner, as expression of GFP-Rab5 accelerated focal adhesion disassembly in nocodazole-synchronized cells, whereas pharmacological inhibition of calpain2 with N-acetyl-Leu-Leu-Met prevented both focal adhesion disassembly and cell migration induced by Rab5. In summary, these data uncover Rab5 as a novel regulator of calpain2 activity and focal adhesion proteolysis leading to cell migration.     

Focal adhesion kinase is activated by microtubule‐depolymerizing agents and regulates membrane blebbing in human endothelial cells

Microtubule‐depolymerizing agents can selectively disrupt tumor vessels via inducing endothelial membrane blebbing. However, the mechanism regulating blebbing is largely unknown. IMB5046 is a newly discovered microtubule‐depolymerizing agent. Here, the functions of focal adhesion kinase (FAK) during IMB5046‐induced blebbing and the relevant mechanism are studied. We found that IMB5046 induced membrane blebbing and reassembly of focal adhesions in human vascular endothelial cells. Both FAK inhibitor and knock‐down expression of FAK inhibited IMB5046‐induced blebbing. Mechanism study revealed that IMB5046 induced the activation of FAK via GEF‐H1/ Rho/ ROCK/ MLC2 pathway. cRGD peptide, a ligand of integrin, also blocked IMB5046‐induced blebbing. After activation, FAK further promoted the phosphorylation of MLC2. This positive feedback loop caused more intensive actomyosin contraction and continuous membrane blebbing. FAK inhibitor blocked membrane blebbing via inhibiting actomyosin contraction, and stimulated stress fibre formation via promoting the phosphorylation of HSP27. Conclusively, these results demonstrate that FAK is a molecular switch controlling endothelial blebbing and stress fibre formation. Our study provides a new molecular mechanism for microtubule‐depolymerizing agents to be used as vascular disrupting agents.     

Integrin adhesions

Cell migration requires the coordination of adhesion site assembly and turnover. Canonical models for nascent adhesion formation postulate that integrin binding to extracellular matrix (ECM) proteins results in the rapid recruitment of cytoskeletal proteins such as talin and paxillin to integrin cytoplasmic domains. It is thought that integrin-talin clusters recruit and activate tyrosine kinases such as focal adhesion kinase (FAK). However, the molecular connections of this linkage remain unresolved. Our recent findings support an alternative model whereby FAK recruits talin to new sites of β1 integrin-mediated adhesion in mouse embryonic fibroblasts and human ovarian carcinoma cells. This is dependent on a direct binding interaction between FAK and talin and occurs independently of direct talin binding to β1 integrin. Herein, we discuss differences between nascent and mature adhesions, interactions between FAK, talin and paxillin, possible mechanisms of FAK activation and how this FAK-talin complex may function to promote cell motility through increased adhesion turnover.     

CXCR4 regulates migration of lung alveolar epithelial cells through activation of Rac1 and matrix metalloproteinase-2

Restoration of the epithelial barrier following acute lung injury is critical for recovery of lung homeostasis. After injury, alveolar type II epithelial (ATII) cells spread and migrate to cover the denuded surface and, eventually, proliferate and differentiate into type I cells. The chemokine CXCL12, also known as stromal cell-derived factor 1α, has well-recognized roles in organogenesis, hematopoiesis, and immune responses through its binding to the chemokine receptor CXCR4. While CXCL12/CXCR4 signaling is known to be important in immune cell migration, the role of this chemokine-receptor interaction has not been studied in alveolar epithelial repair mechanisms. In this study, we demonstrated that secretion of CXCL12 was increased in the bronchoalveolar lavage of rats ventilated with an injurious tidal volume (25 ml/kg). We also found that CXCL12 secretion was increased by primary rat ATII cells and a mouse alveolar epithelial (MLE12) cell line following scratch wounding and that both types of cells express CXCR4. CXCL12 significantly increased ATII cell migration in a scratch-wound assay. When we treated cells with a specific antagonist for CXCR4, AMD-3100, cell migration was significantly inhibited. Knockdown of CXCR4 by short hairpin RNA (shRNA) caused decreased cell migration compared with cells expressing a nonspecific shRNA. Treatment with AMD-3100 decreased matrix metalloproteinase-14 expression, increased tissue inhibitor of metalloproteinase-3 expression, decreased matrix metalloproteinase-2 activity, and prevented CXCL12-induced Rac1 activation. Similar results were obtained with shRNA knockdown of CXCR4. These findings may help identify a therapeutic target for augmenting epithelial repair following acute lung injury.     

PPARδ activation inhibits angiotensin II induced cardiomyocyte hypertrophy by suppressing intracellular Ca2+ signaling pathway

Peroxisome proliferator‐activated receptors δ (PPARδ) is known to be expressed ubiquitously, and the predominant PPAR subtype of cardiac cells. However, relatively less is known regarding the role of PPARδ in cardiac cells except that PPARδ ligand treatment protects cardiac hypertrophy by inhibiting NF‐κB activation. Thus, in the present study, we examined the effect of selective PPARδ ligand L‐165041 on angiotensin II (AngII) induced cardiac hypertrophy and its underlying mechanism using cardiomyocyte. According to our data, L‐165041 (10 µM) inhibited AngII‐induced [³H] leucine incorporation, induction of the fetal gene atrial natriuretic factor (ANF) and increase of cardiomyocyte size. Previous studies have implicated the activation of focal adhesion kinase (FAK) in the progress of cardiomyocyte hypertrophy. L‐165041 pretreatment significantly inhibited AngII‐induced intracellular Ca²⁺ increase and subsequent phosphorylation of FAK. Further experiment using Ca²⁺ ionophore A23187 confirmed that Ca²⁺ induced FAK phosphorylation, and this was also blocked by L‐165041 pretreatment. In addition, overexpression of PPARδ using adenovirus significantly inhibited AngII‐induced intracellular Ca²⁺ increase and FAK expression, while PPARδ siRNA treatment abolished the effect of L‐165041. These data indicate that PPARδ ligand L‐165041 inhibits AngII induced cardiac hypertrophy by suppressing intracellular Ca²⁺/FAK/ERK signaling pathway in a PPARδ dependent mechanism. J. Cell. Biochem. 106: 823–834, 2009. © 2009 Wiley‐Liss, Inc.     

Identification of a bipartite focal adhesion localization signal in RhoU/Wrch-1, a Rho family GTPase that regulates cell adhesion and migration

Background information. Rho GTPases are important regulators of cytoskeleton dynamics and cell adhesion. RhoU/Wrch‐1 is a Rho GTPase which shares sequence similarities with Rac1 and Cdc42 (cell division cycle 42), but has also extended N‐ and C‐terminal domains. The N‐terminal extension promotes binding to SH3 (Src homology 3)‐domain‐containing adaptors, whereas the C‐terminal extension mediates membrane targeting through palmitoylation of its non‐conventional CAAX box. RhoU/Wrch‐1 possesses transforming activity, which is negatively regulated by its N‐terminal extension and depends on palmitoylation. Results. In the present study, we have shown that RhoU is localized to podosomes in osteoclasts and c‐Src‐expressing cells, and to focal adhesions of HeLa cells and fibroblasts. The N‐terminal extension and the palmitoylation site were dispensable, whereas the C‐terminal extension and effector binding loop were critical for RhoU targeting to focal adhesions. Moreover, the number of focal adhesions was reduced and their distribution changed upon expression of activated RhoU. Conversely, RhoU silencing increased the number of focal adhesions. As RhoU was only transiently associated with adhesion structures, this suggests that RhoU may modify adhesion turnover and cell migration rate. Indeed, we found that migration distances were increased in cells expressing activated RhoU and decreased when RhoU was knocked‐down. Conclusions. Our data indicate that RhoU localizes to adhesion structures, regulates their number and distribution and increases cell motility. It also suggests that the RhoU effector binding and C‐terminal domains are critical for these functions.