Dual specificity kinase DYRK3 regulates cell migration by influencing the stability of protrusions.

Plasma membrane-associated platforms (PMAPs) form at specific sites of plasma membrane by scaffolds including ERC1 and Liprin-α1. We identify a mechanism regulating PMAPs assembly, with consequences on motility/invasion. Silencing Ser/Thr kinase DYRK3 in invasive breast cancer cells inhibits their motility and invasive capacity. Similar effects on motility were observed by increasing DYRK3 levels, while kinase-dead DYRK3 had limited effects. DYRK3 overexpression inhibits PMAPs formation and has negative effects on stability of lamellipodia and adhesions in migrating cells. Liprin-α1 depletion results in unstable lamellipodia and impaired cell motility. DYRK3 causes increased Liprin-α1 phosphorylation. Increasing levels of Liprin-α1 rescue the inhibitory effects of DYRK3 on cell spreading, suggesting that an equilibrium between Liprin-α1 and DYRK3 levels is required for lamellipodia stability and tumor cell motility. Our results show that DYRK3 is relevant to tumor cell motility, and identify a PMAP target of the kinase, highlighting a new mechanism regulating cell edge dynamics.

Interfering with the ERC1-LL5ß interaction disrupts plasma membrane-Associated platforms and affects tumor cell motility.

Cell migration requires a complex array of molecular events to promote protrusion at the front of motile cells. The scaffold protein LL5ß interacts with the scaffold ERC1, and recruits it at plasma membrane-associated platforms that form at the front of migrating tumor cells. LL5 and ERC1 proteins support protrusion during migration as shown by the finding that depletion of either endogenous protein impairs tumor cell motility and invasion. In this study we have tested the hypothesis that interfering with the interaction between LL5ß and ERC1 may be used to interfere with the function of the endogenous proteins to inhibit tumor cell migration. For this, we identified ERC1(270-370) and LL5ß(381-510) as minimal fragments required for the direct interaction between the two proteins. The biochemical characterization demonstrated that the specific regions of the two proteins, including predicted intrinsically disordered regions, are implicated in a reversible, high affinity direct heterotypic interaction. NMR spectroscopy further confirmed the disordered nature of the two fragments and also support the occurrence of interaction between them. We tested if the LL5ß protein fragment interferes with the formation of the complex between the two full-length proteins. Coimmunoprecipitation experiments showed that LL5ß(381-510) hampers the formation of the complex in cells. Moreover, expression of either fragment is able to specifically delocalize endogenous ERC1 from the edge of migrating MDA-MB-231 tumor cells. Coimmunoprecipitation experiments show that the ERC1-binding fragment of LL5ß interacts with endogenous ERC1 and interferes with the binding of endogenous ERC1 to full length LL5ß. Expression of LL5ß(381-510) affects tumor cell motility with a reduction in the density of invadopodia and inhibits transwell invasion. These results provide a proof of principle that interfering with heterotypic intermolecular interactions between components of plasma membrane-associated platforms forming at the front of tumor cells may represent a new approach to inhibit cell invasion.

A functional interaction between liprin-α1 and B56γ regulatory subunit of protein phosphatase 2A supports tumor cell motility.

Scaffold liprin-α1 is required to assemble dynamic plasma membrane-associated platforms (PMAPs) at the front of migrating breast cancer cells, to promote protrusion and invasion. We show that the N-terminal region of liprin-α1 contains an LxxIxE motif interacting with B56 regulatory subunits of serine/threonine protein phosphatase 2A (PP2A). The specific interaction of B56γ with liprin-α1 requires an intact motif, since two point mutations strongly reduce the interaction. B56γ mediates the interaction of liprin-α1 with the heterotrimeric PP2A holoenzyme. Most B56γ protein is recovered in the cytosolic fraction of invasive MDA-MB-231 breast cancer cells, where B56γ is complexed with liprin-α1. While mutation of the short linear motif (SLiM) does not affect localization of liprin-α1 to PMAPs, localization of B56γ at these sites specifically requires liprin-α1. Silencing of B56γ or liprin-α1 inhibits to similar extent cell spreading on extracellular matrix, invasion, motility and lamellipodia dynamics in migrating MDA-MB-231 cells, suggesting that B56γ/PP2A is a novel component of the PMAPs machinery regulating tumor cell motility. In this direction, inhibition of cell spreading by silencing liprin-α1 is not rescued by expression of B56γ binding-defective liprin-α1 mutant. We propose that liprin-α1-mediated recruitment of PP2A via B56γ regulates cell motility by controlling protrusion in migrating MDA-MB-231 cells.

Liprins in oncogenic signaling and cancer cell adhesion.

Liprins are a multifunctional family of scaffold proteins, identified by their involvement in several important neuronal functions related to signaling and organization of synaptic structures. More recently, the knowledge on the liprin family has expanded from neuronal functions to processes relevant to cancer progression, including cell adhesion, cell motility, cancer cell invasion, and signaling. These proteins consist of regions, which by prediction are intrinsically disordered, and may be involved in the assembly of supramolecular structures relevant for their functions. This review summarizes the current understanding of the functions of liprins in different cellular processes, with special emphasis on liprins in tumor progression. The available data indicate that liprins may be potential biomarkers for cancer progression and may have therapeutic importance.

Dysregulation of myelin synthesis and actomyosin function underlies aberrant myelin in CMT4B1 neuropathy.

Charcot-Marie-Tooth type 4B1 (CMT4B1) is a severe autosomal recessive demyelinating neuropathy with childhood onset, caused by loss-of-function mutations in the myotubularin-related 2 (MTMR2) gene. MTMR2 is a ubiquitously expressed catalytically active 3-phosphatase, which in vitro dephosphorylates the 3-phosphoinositides PtdIns3P and PtdIns(3,5)P2, with a preference for PtdIns(3,5)P2 A hallmark of CMT4B1 neuropathy are redundant loops of myelin in the nerve termed myelin outfoldings, which can be considered the consequence of altered growth of myelinated fibers during postnatal development. How MTMR2 loss and the resulting imbalance of 3'-phosphoinositides cause CMT4B1 is unknown. Here we show that MTMR2 by regulating PtdIns(3,5)P2 levels coordinates mTORC1-dependent myelin synthesis and RhoA/myosin II-dependent cytoskeletal dynamics to promote myelin membrane expansion and longitudinal myelin growth. Consistent with this, pharmacological inhibition of PtdIns(3,5)P2 synthesis or mTORC1/RhoA signaling ameliorates CMT4B1 phenotypes. Our data reveal a crucial role for MTMR2-regulated lipid turnover to titrate mTORC1 and RhoA signaling thereby controlling myelin growth.

The ERC1 scaffold protein implicated in cell motility drives the assembly of a liquid phase.

Several cellular processes depend on networks of proteins assembled at specific sites near the plasma membrane. Scaffold proteins assemble these networks by recruiting relevant molecules. The scaffold protein ERC1/ELKS and its partners promote cell migration and invasion, and assemble into dynamic networks at the protruding edge of cells. Here by electron microscopy and single molecule analysis we identify ERC1 as an extended flexible dimer. We found that ERC1 scaffolds form cytoplasmic condensates with a behavior that is consistent with liquid phases that are modulated by a predicted disordered region of ERC1. These condensates specifically host partners of a network relevant to cell motility, including liprin-α1, which was unnecessary for the formation of condensates, but influenced their dynamic behavior. Phase separation at specific sites of the cell periphery may represent an elegant mechanism to control the assembly and turnover of dynamic scaffolds needed for the spatial localization and processing of molecules.

The Rac3 GTPase in Neuronal Development, Neurodevelopmental Disorders, and Cancer.

Rho family small guanosine triphosphatases (GTPases) are important regulators of the cytoskeleton, and are critical in many aspects of cellular and developmental biology, as well as in pathological processes such as intellectual disability and cancer. Of the three members of the family, Rac3 has a more restricted expression in normal tissues compared to the ubiquitous member of the family, Rac1. The Rac3 polypeptide is highly similar to Rac1, and orthologues of the gene for Rac3 have been found only in vertebrates, indicating the late appearance of this gene during evolution. Increasing evidence over the past few years indicates that Rac3 plays an important role in neuronal development and in tumor progression, with specificities that distinguish the functions of Rac3 from the established functions of Rac1 in these processes. Here, results highlighting the importance of Rac3 in distinct aspects of neuronal development and tumor cell biology are presented, in support of the non-redundant role of different members of the two Rac GTPases in physiological and pathological processes.

Identification of a membrane-less compartment regulating invadosome function and motility.

Depletion of liprin-α1, ERC1 or LL5 scaffolds inhibits extracellular matrix degradation by invasive cells. These proteins co-accumulate near invadosomes in NIH-Src cells, identifying a novel invadosome-associated compartment distinct from the core and adhesion ring of invadosomes. Depletion of either protein perturbs the organization of invadosomes without influencing the recruitment of MT1-MMP metalloprotease. Liprin-α1 is not required for de novo formation of invadosomes after their disassembly by microtubules and Src inhibitors, while its depletion inhibits invadosome motility, thus affecting matrix degradation. Fluorescence recovery after photobleaching shows that the invadosome-associated compartment is dynamic, while correlative light immunoelectron microscopy identifies bona fide membrane-free invadosome-associated regions enriched in liprin-α1, which is virtually excluded from the invadosome core. The results indicate that liprin-α1, LL5 and ERC1 define a novel dynamic membrane-less compartment that regulates matrix degradation by affecting invadosome motility.

Liprin-α1 and ERC1 control cell edge dynamics by promoting focal adhesion turnover.

Liprin-α1 and ERC1 are interacting scaffold proteins regulating the motility of normal and tumor cells. They act as part of plasma membrane-associated platforms at the edge of motile cells to promote protrusion by largely unknown mechanisms. Here we identify an amino-terminal region of the liprin-α1 protein (liprin-N) that is sufficient and necessary for the interaction with other liprin-α1 molecules. Similar to liprin-α1 or ERC1 silencing, expression of the liprin-N negatively affects tumor cell motility and extracellular matrix invasion, acting as a dominant negative by interacting with endogenous liprin-α1 and causing the displacement of the endogenous ERC1 protein from the cell edge. Interfering with the localization of ERC1 at the cell edge inhibits the disassembly of focal adhesions, impairing protrusion. Liprin-α1 and ERC1 proteins colocalize with active integrin ß1 clusters distinct from those colocalizing with cytoplasmic focal adhesion proteins, and influence the localization of peripheral Rab7-positive endosomes. We propose that liprin-α1 and ERC1 promote protrusion by displacing cytoplasmic adhesion components to favour active integrin internalization into Rab7-positive endosomes.

Role of Liprins in the Regulation of Tumor Cell Motility and Invasion.

Invasion leading to the formation of metastasis is one of the hallmarks of cancer. Analysis of different human cancers has led to the identification of the PPFIA1 gene encoding the protein liprin-α1, a possible player in cancer. The PPFIA1 gene is amplified in malignant tumors, including about 20% of breast cancers. Also the liprin-α1 protein is found overexpressed in tumors. Liprin-α1 belongs to the liprin family of cytosolic scaffold proteins that includes four liprin-α, two liprin-ß members, and liprin-γ/kazrinE. In this review we will discuss the available evidence on the role of different members of the liprin family in distinct aspects of tumor cell migration and invasion. Evidence from in vitro studies indicates that the widely expressed liprin-α1 protein regulates the migration and invasion of human breast cancer cells. Liprin-α1 affects cell migration and invasion by regulating the organization of lamellipodia and invadopodia, two structures relevant to cell invasion. In the cell liprin-α1 forms a complex with liprin-ß1, ERC1/ELKS and LL5 proteins, which localizes at the front of migrating cells and positively regulates lamellipodia stability, and integrin-mediated focal adhesions. On the other hand, liprin-ß2 appears to play a role as tumor suppressor by inhibiting breast cancer cell motility and invasion. The available data indicate that liprins are central players in the regulation of tumor cell invasion, therefore representing interesting targets for anti-metastatic therapy.

Effects of the scaffold proteins liprin-α1, ß1 and ß2 on invasion by breast cancer cells.

BACKGROUND INFORMATION: The expression of the scaffold protein liprin-α1 is upregulated in human breast cancer. This protein is part of a molecular network that is important for tumour cell invasion in vitro. Liprin-α1 promotes invasion by supporting the protrusive activity at the leading edge of the migrating tumour cell and the degradation of the extracellular matrix by invadopodia. In this study, we have addressed the role of liprin-α1 in the invasive process in vivo and of liprin-proteins in tumor cell motility. RESULTS: The human tumour cell line MDA-MB-231 expresses liprin-α1 and is able to promote the formation of metastasis in mice. Liprin-α proteins may hetero-oligomerize with the members of the subfamily of the liprin-ß adaptor proteins. Analysis of the role of liprin-ß1 and liprin-ß2 has shown that while liprin-ß1 contributes positively to tumour cell motility in vitro; liprin-ß2 has a negative effect on both cell motility and invasion. Interestingly, we also observed differential effects on the ability of tumour cells to degrade the extracellular matrix, which is required for efficient invasion by tumour cells. In addition, analysis of the formation of lung metastases in vivo revealed that while the overexpression of liprin-α1 in MDA-MB-231 cells did not evidently affect the metastatic process, silencing of the endogenous protein strongly impaired the formation of metastases by two independent invasion assays, without inhibiting the growth of primary tumours. CONCLUSIONS: Our data support an important role of distinct liprin family members in the regulation of tumour cell invasion, highlighting pro-invasive and anti-invasive effects by liprin-α1 and liprin-ß2, respectively. SIGNIFICANCE: Our results indicate the importance of liprins in breast cancer cell invasion, and are expected to lead to future investigations on the mechanisms underlying the effects of distinct liprin proteins in different processes linked to tumor cell migration and invasion.

Liprin-α1, ERC1 and LL5 define polarized and dynamic structures that are implicated in cell migration.

Cell migration during development and metastatic invasion requires the coordination of actin and adhesion dynamics to promote protrusive activity at the front of the cell. The knowledge of the molecular mechanisms required to achieve such coordination is fragmentary. Here, we identify a new functional complex that drives cell motility. ERC1a (an isoform of ERC1) and the LL5 proteins LL5α and LL5ß (encoded by PHLDB1 and PHLDB2, respectively) are required, together with liprin-α1, for effective migration and tumor cell invasion, and do so by stabilizing the protrusive activity at the cell front. Depletion of either protein negatively affects invasion, migration on extracellular matrix, lamellipodial persistence and the internalization of active integrin ß1 receptors needed for adhesion turnover at the front of the cell. Liprin-α1, ERC1a and LL5 also define new highly polarized and dynamic cytoplasmic structures uniquely localized near the protruding cell edge. Our results indicate that the functional complex and the associated structures described here represent an important mechanism to drive tumor cell migration.

Identification of two tyrosine residues required for the intramolecular mechanism implicated in GIT1 activation.

GIT1 is an ArfGAP and scaffolding protein regulating cell adhesion and migration. The multidomain structure of GIT1 allows the interaction with several partners. Binding of GIT1 to some of its partners requires activation of the GIT1 polypeptide. Our previous studies indicated that binding of paxillin to GIT1 is enhanced by release of an intramolecular interaction between the amino-terminal and carboxy-terminal portions that keeps the protein in a binding-incompetent state. Here we have addressed the mechanism mediating this intramolecular inhibitory mechanism by testing the effects of the mutation of several formerly identified GIT1 phosphorylation sites on the binding to paxillin. We have identified two tyrosines at positions 246 and 293 of the human GIT1 polypeptide that are needed to keep the protein in the inactive conformation. Interestingly, mutation of these residues to phenylalanine did not affect binding to paxillin, while mutation to either alanine or glutamic acid enhanced binding to paxillin, without affecting the constitutive binding to the Rac/Cdc42 exchange factor ßPIX. The involvement of the two tyrosine residues in the intramolecular interaction was supported by reconstitution experiments showing that these residues are important for the binding between the amino-terminal fragment and carboxy-terminal portions of GIT1. Either GIT1 or GIT1-N tyrosine phosphorylation by Src and pervanadate treatment to inhibit protein tyrosine phosphatases did not affect the intramolecular binding between the amino- and carboxy-terminal fragments, nor the binding of GIT1 to paxillin. Mutations increasing the binding of GIT1 to paxillin positively affected cell motility, measured both by transwell migration and wound healing assays. Altogether these results show that tyrosines 246 and 293 of GIT1 are required for the intramolecular inhibitory mechanism that prevents the binding of GIT1 to paxillin. The data also suggest that tyrosine phosphorylation may not be sufficient to release the intramolecular interaction that keeps GIT1 in the inactive conformation.

Biochemical and functional characterization of the interaction between liprin-α1 and GIT1: implications for the regulation of cell motility.

We have previously identified the scaffold protein liprin-α1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-α1 may interact with different proteins, and the functional significance of these interactions in the regulation of cell motility is poorly known. Here we have addressed the involvement of the liprin-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration. GIT1 depletion inhibited spreading by affecting the lamellipodia, and prevented liprin-α1-enhanced spreading. Conversely inhibition of the formation of the liprin-α1-GIT complex by expression of liprin-ΔCC3 could still enhance spreading, although to a lesser extent compared to full length liprin-α1. No cumulative effects were observed after depletion of both liprin-α1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading. Our data suggest that liprin-α1 may compete with paxillin for binding to GIT1, while binding of ßPIX to GIT1 was unaffected by the presence of liprin-α1. Interestingly, GIT and liprin-α1 reciprocally regulated their subcellular localization, since liprin-α1 overexpression, but not the GIT binding-defective liprin-ΔCC3 mutant, affected the localization of endogenous GIT at peripheral and mature central focal adhesions, while the expression of a truncated, active form of GIT1 enhanced the localization of endogenous liprin-α1 at the edge of spreading cells. Moreover, GIT1 was required for liprin-α1-enhanced haptotatic migration, although the direct interaction between liprin-α1 and GIT1 was not needed. Our findings show that the functional interaction between liprin-α1 and GIT1 cooperate in the regulation of integrin-dependent cell spreading and motility on extracellular matrix. These findings and the possible competition of liprin-α1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-α1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.

Function of liprins in cell motility.

Liprins have been known for years to play an essential role in setting up functional synapses in the nervous system. On the other hand, these proteins had been first identified in non-neuronal cells as multivalent proteins that may affect the integrin-mediated interactions of the cells with extracellular matrix ligands. Although the research on the function of liprins in non-neuronal cells has been quiescent for several years, a number of recent findings are putting them back on stage again as important players also in the regulation of non-neuronal cell motility, and possibly of tumor cell behavior. The aim of this review is to highlight the findings supporting the importance of liprins as central regulators of cell adhesion and motility, making them an interesting family of proteins to be considered for future studies on the mechanisms regulating cell migration.

The GIT-PIX complexes regulate the chemotactic response of rat basophilic leukaemia cells.

BACKGROUND INFORMATION: Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT-PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G-protein-coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl-Met-Leu-Phe) peptide, in a rat basophilic leukaemia RBL-2H3 cell line stably expressing an HA (haemagglutinin)-tagged receptor for the fMLP peptide. RESULTS: Our analysis shows that RBL cells stably transfected with the chemoattractant receptor expressed both GIT1-PIX and GIT2-PIX endogenous complexes. We have used silencing of the different members of the complex by small interfering RNAs to study the effects on a number of events linked to agonist-induced cell migration. We found that cell adhesion was not affected by depletion of any of the proteins of the GIT complex, whereas agonist-enhanced cell spreading was inhibited. Analysis of agonist-stimulated haptotactic cell migration indicated a specific positive effect of GIT1 depletion on trans-well migration. The internalization of the formyl-peptide receptor was also inhibited by depletion of GIT1 and GIT2. The effects of the GIT complexes on trafficking of the receptors was confirmed by an antibody-enhanced agonist-induced internalization assay, showing that depletion of PIX, GIT1 or GIT2 protein caused decreased perinuclear accumulation of internalized receptors. CONCLUSIONS: Our results show that endogenous GIT complexes are involved in the regulation of chemoattractant-induced cell motility and receptor trafficking, and support previous findings indicating an important function of the GIT complexes in the regulation of different G-protein-coupled receptors. Our results also indicate that endogenous GIT1 and GIT2 regulate distinct subsets of agonist-induced responses and suggest a possible functional link between the control of receptor trafficking and the regulation of cell motility by GIT proteins.

Generation of rac3 null mutant mice: role of Rac3 in Bcr/Abl-caused lymphoblastic leukemia.

Numerous studies indirectly implicate Rac GTPases in cancer. To investigate if Rac3 contributes to normal or malignant cell function, we generated rac3 null mutants through gene targeting. These mice were viable, fertile, and lacked an obvious external phenotype. This shows Rac3 function is dispensable for embryonic development. Bcr/Abl is a deregulated tyrosine kinase that causes chronic myelogenous leukemia and Ph-positive acute lymphoblastic leukemia in humans. Vav1, a hematopoiesis-specific exchange factor for Rac, was constitutively tyrosine phosphorylated in primary lymphomas from Bcr/Abl P190 transgenic mice, suggesting inappropriate Rac activation. rac3 is expressed in these malignant hematopoietic cells. Using lysates from BCR/ABL transgenic mice that express or lack rac3, we detected the presence of activated Rac3 but not Rac1 or Rac2 in the malignant precursor B-lineage lymphoblasts. In addition, in female P190 BCR/ABL transgenic mice, lack of rac3 was associated with a longer average survival. These data are the first to directly show a stimulatory role for Rac in leukemia in vivo. Moreover, our data suggest that interference with Rac3 activity, for example, by using geranyl-geranyltransferase inhibitors, may provide a positive clinical benefit for patients with Ph-positive acute lymphoblastic leukemia.

Analysis of the subcellular distribution of avian p95-APP2, an ARF-GAP orthologous to mammalian paxillin kinase linker.

We describe here the identification and characterization of avian p95-APP2, a multi-domain protein of a recently identified family of ADP-ribosylation factor (ARF)-GTPase-activating proteins (GAPs) including mammalian G protein-coupled receptor kinases (GRK)-interactor 1 (GIT1), paxillin kinase linker (PKL), and GIT2, as well as avian p95-APP1. The p95-APP2 is eluted from Rac-GTP-gamma-S, but not from Rac-GDP-beta-S columns. As other members of the family, p95-APP2 has binding regions for the focal adhesion protein paxillin, and for the Rac exchanging factor PIX. Sequence comparison indicates that p95-APP2 is the avian orthologue of mammalian PKL. Expression studies showed a largely diffuse distribution of the full length p95-APP2, without evident effects on cell morphology. We observed a dramatic difference between the localization of the amino-terminal portion of the protein, including the ARF-GAP domain and the three ankyrin repeats, and the carboxy-terminal portion including the paxillin-binding site. Moreover, the expression of truncated carboxy-terminal polypeptides including both the PIX- and paxillin-binding regions leads to a marked localization of the protein together with paxillin at large vesicles. Comparison of the expression of corresponding ARF-GAP-deficient constructs from p95-APP2 and p95-APP1 shows their distribution at distinct endocytic compartments. Altogether, these data support a role of distinct members of this family of ARF-GAPs in the regulation of different steps of membrane traffic during cell motility, and suggest that p95-APP2 may shuttle between an intracellular compartment and the cell periphery, although, further work will be needed to address this point.