Vinculin Controls PTEN Protein Level by Maintaining the Interaction of the Adherens Junction Protein β-Catenin with the Scaffolding Protein MAGI-2

PTEN is a frequently mutated tumor suppressor in malignancies. Interestingly, some malignancies exhibit undetectable PTEN protein without mutations or loss of PTEN mRNA. The cause(s) for this reduction in PTEN is unknown. Cancer cells frequently exhibit loss of cadherin, beta-catenin, alpha-catenin and/or vinculin, key elements of adherens junctions. Here we show that F9 vinculin-null (vin(-/-)) cells lack PTEN protein despite normal PTEN mRNA levels. Their PTEN protein expression was restored by transfection with vinculin or by inhibition of PTEN degradation. F9 vin(-/-) cells express PTEN protein upon transfection with a vinculin fragment (amino acids 243-1066) that is capable of interacting with alpha-catenin but unable to target into focal adhesions. On the other hand, disruption of adherens junctions with an E-cadherin blocking antibody reduced PTEN protein to undetectable levels in wild-type F9 cells. PTEN protein levels were restored in F9 vin(-/-) cells upon transfection with an E-cadherin-alpha-catenin fusion protein, which targets into adherens junctions and interacts with beta-catenin in F9 vin(-/-) cells. beta-Catenin is known to interact with MAGI-2. MAGI-2 interaction with PTEN in the cell membrane is known to prevent PTEN protein degradation. Thus, MAGI-2 overexpression in F9 vin(-/-) cells restored PTEN protein levels. Moreover, expression of vinculin mutants that reinstated the disrupted interactions of beta-catenin with MAGI-2 in F9 vin(-/-) cells also restored PTEN protein levels. These studies indicate that PTEN protein levels are dependent on the maintenance of beta-catenin-MAGI-2 interaction, in which vinculin plays a critical role

RhoA GTPase Activation by TLR2 and TLR3 Ligands: Connecting via Src to NF- B

Rho GTPases are essential regulators of signaling networks emanating from many receptors involved in innate or adaptive immunity. The Rho family member RhoA controls cytoskeletal processes as well as the activity of transcription factors such as NF-κB, C/EBP and serum response factor. The multifaceted host cell activation triggered by Toll-like receptors (TLRs) in response to soluble and particulate microbial structures includes rapid stimulation of RhoA activity. RhoA acts downstream of TLR2 in HEK-TLR2 and monocytic THP-1 cells, but the signaling pathway connecting TLR2 and RhoA is still unknown. It is also not clear if RhoA activation is dependent on a certain TLR adapter. Using lung epithelial cells, we demonstrate TLR2- and TLR3-triggered recruitment and activation of RhoA at receptor-proximal cellular compartments. RhoA activity was dependent on TLR-mediated stimulation of Src family kinases. Both Src family kinases and RhoA were required for NF-κB activation, while RhoA was dispensable for type I interferon generation. These results suggest that RhoA plays a role downstream of MyD88-dependent and -independent TLR signaling and acts as a molecular switch downstream of TLR-Src initiated pathways

Positive feedback between Cdc42 activity and H + efflux by the Na-H exchanger NHE1 for polarity of migrating cells

A fundamental feature of cell polarity in response to spatial cues is asymmetric amplification of molecules generated by positive feedback signaling. We report a positive feedback loop between the guanosine triphosphatase Cdc42, a central determinant in eukaryotic cell polarity, and H+ efflux by Na-H+ exchanger 1 (NHE1), which is necessary at the front of migrating cells for polarity and directional motility. In response to migratory cues, Cdc42 is not activated in fibroblasts expressing a mutant NHE1 that lacks H+ efflux, and wild-type NHE1 is not activated in fibroblasts expressing mutationally inactive Cdc42-N17. H+ efflux by NHE1 is not necessary for release of Cdc42–guanosine diphosphate (GDP) from Rho GDP dissociation inhibitor or for the membrane recruitment of Cdc42 but is required for GTP binding by Cdc42 catalyzed by a guanine nucleotide exchange factor (GEF). Data indicate that GEF binding to phosphotidylinositol 4,5–bisphosphate is pH dependent, suggesting a mechanism for how H+ efflux by NHE1 promotes Cdc42 activity to generate a positive feedback signal necessary for polarity in migrating cells

GEF-H1 Modulates Localized RhoA Activation during Cytokinesis under the Control of Mitotic Kinases

Formation of the mitotic cleavage furrow is dependent upon both microtubules and activity of the small GTPase RhoA. GEF-H1 is a microtubule-regulated exchange factor that couples microtubule dynamics to RhoA activation. GEF-H1 localized to the mitotic apparatus in HeLa cells, particularly at the tips of cortical microtubules and the midbody, and perturbation of GEF-H1 function induced mitotic aberrations, including asymmetric furrowing, membrane blebbing, and impaired cytokinesis. The mitotic kinases Aurora A/B and Cdk1/Cyclin B phosphorylate GEF-H1, thereby inhibiting GEF-H1 catalytic activity. Dephosphorylation of GEF-H1 occurs just prior to cytokinesis, accompanied by GEF-H1-dependent GTP-loading on RhoA. Using a live cell biosensor, we demonstrate distinct roles for GEF-H1 and Ect2 in regulating Rho activity in the cleavage furrow, with GEF-H1 catalyzing Rho activation in response to Ect2-dependent localization and initiation of cell cleavage. Our results identify a GEF-H1-dependent mechanism to modulate localized RhoA activation during cytokinesis under the control of mitotic kinases

Coordination of Rho GTPase activities during cell protrusion

The GTPases Rac1, RhoA and Cdc42 act in concert to control cytoskeleton dynamics1-3. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells4-7 and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac18, and Rac1 and RhoA are mutually inhibitory9-12. However, their spatiotemporal coordination, at the seconds and single micron dimensions typical of individual protrusion events, remains unknown. Here, we examine GTPase coordination both through simultaneous visualization of two GTPase biosensors and using a “computational multiplexing” approach capable of defining the relationships between multiple protein activities visualized in separate experiments. We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 μm behind the edge with a delay of 40 sec. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA plays a role in the initial events of protrusion, while Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions

PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK−/− or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK–p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility

An excitable Rho GTPase signaling network generates dynamic subcellular contraction patterns.

Rho GTPase-based signaling networks control cellular dynamics by coordinating protrusions and retractions in space and time. Here, we reveal a signaling network that generates pulses and propagating waves of cell contractions. These dynamic patterns emerge via self-organization from an activator-inhibitor network, in which the small GTPase Rho amplifies its activity by recruiting its activator, the guanine nucleotide exchange factor GEF-H1. Rho also inhibits itself by local recruitment of actomyosin and the associated RhoGAP Myo9b. This network structure enables spontaneous, self-limiting patterns of subcellular contractility that can explore mechanical cues in the extracellular environment. Indeed, actomyosin pulse frequency in cells is altered by matrix elasticity, showing that coupling of contractility pulses to environmental deformations modulates network dynamics. Thus, our study reveals a mechanism that integrates intracellular biochemical and extracellular mechanical signals into subcellular activity patterns to control cellular contractility dynamics

Charakterisierung von zwei Na + -Phosphat-Kotransportern des Typs NaPi-IIb aus dem Zebrafisch (Brachydanio rerio) und die Regulation durch Antisense-Transkripte

Die Proteinfamilie NaPi-II ist in Vertebraten für den natriumabhängigen Phosphattransport der Epithelzellen in Niere und Darm verantwortlich. Durch Regulation dieses Proteins wird der Phosphattransport kontrolliert und dadurch die Phosphathomöostase aufrechterhalten. Physiologische Unterschiede zwischen Säugern und Fischen fokussierten das Interesse in meiner Diplomarbeit auf den Na+/Phosphat-Kotransporter des Zebrafisches (Brachydanio rerio). Interessanterweise wurden aus Darm und Niere dieses Süßwasserfisches zwei Transkripte isoliert, die für zwei unterschiedliche Na+/Phosphat-Kotransporter der Proteinfamilie NaPi-II kodieren. Die Isoform aus dem Darm wurde vollständig kloniert und kodiert für ein Protein mit 634 Aminosäuren. Die Analyse der Aminosäuresequenz von diesem Klon zeigte, daß sich das Protein in die NaPi-IIb Untergruppe der Transporterfamilie einordnen läßt. Die Nieren-Isoform konnte nur teilweise isoliert werden. Ziel der vorliegenden Arbeit war es, beide NaPi-II Isoformen des Zebrafisches auf molekularer Ebene und funktionell zu charakterisieren und so die physiologische Relevanz dieser Transkripte einzugrenzen. Die 5’-RACE Untersuchungen im Falle der Nieren-Isoform erbrachten lediglich Produkte ohne ein sinnvolles Start-Methionin im NaPi-II Leserahmen. Der homologe Abschnitt des gesamten Transkripts umfaßt 2010 bp und kodiert im NaPi-II Leserahmen für ein Protein mit 633 Aminosäuren. Die erhaltenen Sequenzdaten erlaubten weitere Untersuchungen auf Nukleotid und Proteinebene. Auch die Nieren-Isoform wird demnach der Untergruppe NaPi-IIb zugeordnet. Die Isoformen wurden im Folgenden NaPi-IIb1 (ursprünglich: Darm) und NaPi-IIb2 (ursprünglich: Niere) genannt. Die Gewebeverteilung der mRNA beider Isoformen wurde mit RT-PCR untersucht. Die Darm-Isoform wurde wie erwartet im Darm, in den Augen und in der Niere nachgewiesen. Dagegen konnte die Nieren-Isoform in nahezu allen untersuchten Organen detektiert werden. Mit Antikörpern aus dem 3’-Bereich beider NaPi-IIb Proteine wurden immunhistochemische Untersuchungen an Gewebeschnitten durchgeführt. Beide Proteine werden exprimiert und sind in Epithelzellen des Darms, in Nieren-Tubuli und in Gallengängen (Leber) apikal lokalisiert. Der Nachweis der Expression trotz der RACE-Ergebnisse bei NaPi-IIb2 zeigt, daß möglicherweise eine alternative Spleißvariante des isolierten Transkripts mit einem offenen Leserahmen existiert. In elektrophysiologischen Untersuchungen zeigte die klonierte Isoform NaPi-IIb1 ähnliche Transporteigenschaften wie andere NaPi-II Proteine. Während bei Säugern ein deutlicher Unterschied zwischen typischen NaPi-IIa und NaPi-IIb Transportcharakteristika existiert, zeigt der Transport durch Zebrafisch-NaPi-IIb überlappende funktionellen Eigenschaften mit den Typ IIa Transportern. Es wurden zwei natürliche Antisense-mRNAs des NaPi-IIb1 Transkripts entdeckt und kloniert. Aufgrund des gemeinsamen genomischen Locus besitzen beide Transkripte komplementäre Bereiche zu NaPi-IIb1 mRNA. Die Koinjektion von NaPi-IIb1 mit jeweils einem der Antisense-Transkripte hemmte den Phosphattransport erheblich. Durch Northern-Analysen und Immunhistochemie an Oozyten konnte gezeigt werden, daß diese Beobachtung nicht auf den Abbau der injizierten NaPi-IIb1 cRNA zurückzuführen ist, sondern durch die Hemmung der Proteintranslation hervorgerufen wird. In vivo ergaben RT-PCR Untersuchungen, daß die Gewebeexpression von NaPi-IIb1 mRNA mit dem Vorhandensein seiner Antisense-Transkripte verknüpft ist. In Organen, in welchen Antisense-mRNA detektiert werden konnte, konnte keine NaPi-IIb1 mRNA nachgewiesen werden. Dieser Befund weist auf eine mögliche regulatorische Rolle der Antisense-Transkripte im Zebrafisch hin