Y-27632, an Inhibitor of Rho-Associated Kinases, Prevents Tyrosine Phosphorylation of Focal Adhesion Kinase and Paxillin Induced by Bombesin: Dissociation from Tyrosine Phosphorylation of p130cas

Sinnett‐Smith, James; Lunn, John; Leopoldt, Daniela; Rozengurt, Enrique

doi:10.1006/excr.2001.5219

Cited by 69 publications

(61 citation statements)

References 70 publications

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“…Enlarging on this last point, and as stated above, a growing set of proteins associated with focal adhesions, including the integrins, FAK, and others have been shown to be engaged in a reciprocal regulatory dialog with RhoA in control of cell attachment and cell motility during interphase (Flinn and Ridley, 1996;Arthur et al, 2000;Ren et al, 2000;Schwartz and Shattil, 2000;Sinnett-Smith et al, 2001;Arthur et al, 2002;Danen et al, 2002). FAK (Ϫ/Ϫ) null cells are nonmotile and excessively rounded; however, inhibition of p160ROCK in FAK(Ϫ/Ϫ) cells reverses rounding, implying FAK functions are dependent on RhoA (Chen et al, 2002).…”

Section: Discussionmentioning

confidence: 92%

Deregulation of HEF1 Impairs M-Phase Progression by Disrupting the RhoA Activation Cycle

Dadke

Jarnik

Pugacheva

et al. 2006

MBoC

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The focal adhesion-associated signaling protein HEF1 undergoes a striking relocalization to the spindle at mitosis, but a function for HEF1 in mitotic signaling has not been demonstrated. We here report that overexpression of HEF1 leads to failure of cells to progress through cytokinesis, whereas depletion of HEF1 by small interfering RNA (siRNA) leads to defects earlier in M phase before cleavage furrow formation. These defects can be explained mechanistically by our determination that HEF1 regulates the activation cycle of RhoA. Inactivation of RhoA has long been known to be required for cytokinesis, whereas it has recently been determined that activation of RhoA at the entry to M phase is required for cellular rounding. We find that increased HEF1 sustains RhoA activation, whereas depleted HEF1 by siRNA reduces RhoA activation. Furthermore, we demonstrate that chemical inhibition of RhoA is sufficient to reverse HEF1-dependent cellular arrest at cytokinesis. Finally, we demonstrate that HEF1 associates with the RhoA-GTP exchange factor ECT2, an orthologue of the Drosophila cytokinetic regulator Pebble, providing a direct means for HEF1 control of RhoA. We conclude that HEF1 is a novel component of the cell division control machinery and that HEF1 activity impacts division as well as cell attachment signaling events. INTRODUCTIONAs points of structural linkage between the extracellular matrix (ECM) and the intracellular cytoskeleton, focal adhesions possess a complex function. For example, during migration, cells must rapidly break down and reform adhesions with the ECM, providing force for propulsion (Lauffenburger and Horwitz, 1996). At mitotic entry, cultured cells round up and decrease adhesion to the ECM; at mitotic exit, basal attachments reassemble and contribute to the force generation required for efficient progress through cytokinesis and reentry into G 1 . In interphase cells, the formation of novel focal adhesion-ECM interactions can specify cellular differentiation by activating specific signaling cascades culminating in the induction of differentiationpromoting transcription factors, and in parallel enforce removal from the cell cycle (Boudreau and Bissell, 1998). In many cell types, sustained loss of adhesion is a sufficient stimulus to induce apoptosis (anoikis) (Frisch and Francis, 1994), a surveillance mechanism against cancer, inhibiting the formation of micrometastases. Hence, one frequent effect of oncogenic transformation is the circumvention of the adhesion-viability coupling, leading to acquisition by cancer cells of the ability to grow in an anchorage-independent manner (Schwartz, 1997). Based on these diverse biological roles, there has been considerable research effort directed at elucidating the signaling role of focal adhesion-associated proteins (Schlaepfer et al., 1999).HEF1, p130Cas, and Efs/Sin define the Cas family of proteins Bouton et al., 2001). In interphase cells, Cas proteins predominantly localize to focal adhesions. During initial integrin engagement, induced by cell a...

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Section: Discussionmentioning

confidence: 92%

Deregulation of HEF1 Impairs M-Phase Progression by Disrupting the RhoA Activation Cycle

Dadke

Jarnik

Pugacheva

et al. 2006

MBoC

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“…The structurally unrelated compound Y-27632 was also identified as a selective inhibitor, affecting ROK 200-fold more than PKC (61) and 20-to 30-fold more than other Rho-dependent kinases (24). The Y-27632 compound, like HA-1077, prevented GPCR-mediated stress fiber formation in fibroblasts (51) and recently was shown in T cells to induce disorganization of cortical actin in an identical manner to that of C3 exoenzyme (44).…”

Section: Ang Ii-or Lpa-induced Pyk2 Tyrosine Phosphorylation Is Inhibmentioning

confidence: 88%

“…To disrupt this pathway, we used the compounds 1-(5-isoquinolone sulfonyl)homopiperazine (HA-1077) and Y-27632, two selective, cell-permeable, and structurally distinct ROK inhibitors. HA-1077 was identified as a potent, preferential inhibitor of ROK (11, 38) that has been shown to prevent GPCR-mediated actin stress fiber assembly (51). The structurally unrelated compound Y-27632 was also identified as a selective inhibitor, affecting ROK 200-fold more than PKC (61) and 20-to 30-fold more than other Rho-dependent kinases (24).…”

Section: Ang Ii-or Lpa-induced Pyk2 Tyrosine Phosphorylation Is Inhibmentioning

confidence: 99%

ANG II and LPA induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells: role of Ca²⁺, PKC, and Rho kinase

Wu¹,

Chiu

Rozengurt

2002

American Journal of Physiology-Cell Physiology

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2001.-The G protein-coupled receptor agonists angiotensin II (ANG II) and lysophosphatidic acid (LPA) rapidly induce tyrosine phosphorylation of the cytosolic proline-rich tyrosine kinase 2 (Pyk2) in IEC-18 intestinal epithelial cells. The combined Pyk2 tyrosine phosphorylation induced by phorbol 12,13-dibutyrate, a direct agonist of protein kinase C (PKC), and ionomycin, a Ca 2ϩ ionophore, was equal to that induced by ANG II. Inhibition of either PKC or Ca 2ϩ signaling attenuated the effect of ANG II and LPA, although simultaneous inhibition of both pathways failed to completely abolish Pyk2 tyrosine phosphorylation. Cytochalasin D, which disrupts stress fibers, strongly inhibited the response of Pyk2 to ANG II or LPA. The distinct Rho-associated kinase (ROK) inhibitors HA-1077 and Y-27632, as well as the Rho inhibitor Clostridium botulinum C3 exoenzyme, also significantly attenuated ANG II-and LPA-stimulated Pyk2 tyrosine phosphorylation. Simultaneous inhibition of PKC, Ca 2ϩ , and either actin assembly or ROK completely abolished the Pyk2 response. Together, these results show that ANG II and LPA rapidly induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells via separate Ca 2ϩ -, PKC-, and Rho-mediated pathways.

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“…68,69 Experiments to determine the relative importance of PA SMC versus other cell types in the observed cytoprotective effects of NEP are being developed. In addition, we are expanding the current observations made in mice to the study of relevant human disease states.…”

Section: Discussionmentioning

confidence: 99%

Neprilysin Null Mice Develop Exaggerated Pulmonary Vascular Remodeling in Response to Chronic Hypoxia

Dempsey

Wick

Karoor³

et al. 2009

The American Journal of Pathology

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Neprilysin is a transmembrane metalloendopeptidase that degrades neuropeptides that are important for both growth and contraction. In addition to promoting carcinogenesis, decreased levels of neprilysin increases inflammation and neuroendocrine cell hyperplasia, which may predispose to vascular remodeling. Early pharmacological studies showed a decrease in chronic hypoxic pulmonary hypertension with neprilysin inhibition. We used a genetic approach to test the alternate hypothesis that neprilysin depletion increases chronic hypoxic pulmonary hypertension. Loss of neprilysin had no effect on baseline airway or alveolar wall architecture, vessel density, cardiac function, hematocrit, or other relevant peptidases. Only lung neuroendocrine cell hyperplasia and a subtle neuropeptide imbalance were found. After chronic hypoxia, neprilysin-null mice exhibited exaggerated pulmonary hypertension and striking increases in muscularization of distal vessels. Subtle thickening of proximal media/adventitia not typically seen in mice was also detected. In contrast, adaptive right ventricular hypertrophy was less than anticipated. Hypoxic wild-type pulmonary vessels displayed close temporal and spatial relationships between decreased neprilysin and increased cell growth. Smooth muscle cells from neprilysin-null pulmonary arteries had increased proliferation compared with controls, which was decreased by neprilysin replacement. These data suggest that neprilysin may be protective against chronic hypoxic pulmonary hypertension in the lung, at least in part by attenuating the growth of smooth muscle cells. Lung-targeted strategies to increase neprilysin levels could have therapeutic benefits in the treatment of this disorder. Chronic hypoxic pulmonary hypertension (PHTN) is a major clinical problem, complicating most lung and heart disorders. 1,2 In large animal models of chronic hypoxic PHTN that closely resemble human disease, the earliest pulmonary artery (PA) smooth muscle cell (SMC) proliferative changes occur at the medial/adventitial border. 3 Growth and migration of SMC and myofibroblasts in distal vessels is also a prominent feature. 4,5 These structural changes, together with derangements in vascular tone, are major contributors to the severity of chronic hypoxic PHTN. [1][2][3][4][5][6] However, mechanisms that regulate susceptibility to, and severity of, chronic hypoxic PHTN and vascular remodeling remain poorly understood. Currently available treatments for chronic hypoxic PHTN are also inadequate.

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Y-27632, an Inhibitor of Rho-Associated Kinases, Prevents Tyrosine Phosphorylation of Focal Adhesion Kinase and Paxillin Induced by Bombesin: Dissociation from Tyrosine Phosphorylation of p130cas

Cited by 69 publications

References 70 publications

Deregulation of HEF1 Impairs M-Phase Progression by Disrupting the RhoA Activation Cycle

Deregulation of HEF1 Impairs M-Phase Progression by Disrupting the RhoA Activation Cycle

ANG II and LPA induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells: role of Ca²⁺, PKC, and Rho kinase

Neprilysin Null Mice Develop Exaggerated Pulmonary Vascular Remodeling in Response to Chronic Hypoxia

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Y-27632, an Inhibitor of Rho-Associated Kinases, Prevents Tyrosine Phosphorylation of Focal Adhesion Kinase and Paxillin Induced by Bombesin: Dissociation from Tyrosine Phosphorylation of p130cas

Cited by 69 publications

References 70 publications

Deregulation of HEF1 Impairs M-Phase Progression by Disrupting the RhoA Activation Cycle

Deregulation of HEF1 Impairs M-Phase Progression by Disrupting the RhoA Activation Cycle

ANG II and LPA induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells: role of Ca2+, PKC, and Rho kinase

Neprilysin Null Mice Develop Exaggerated Pulmonary Vascular Remodeling in Response to Chronic Hypoxia

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ANG II and LPA induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells: role of Ca²⁺, PKC, and Rho kinase