scholarly journals The Rho kinases I and II regulate different aspects of myosin II activity

2005 ◽  
Vol 170 (3) ◽  
pp. 443-453 ◽  
Author(s):  
Atsuko Yoneda ◽  
Hinke A.B. Multhaupt ◽  
John R. Couchman
Keyword(s):  
Rho Gtpases ◽  
Myosin Light Chain ◽  
Focal Adhesions ◽  
Lipid Binding ◽  
Smooth Muscle Contractility ◽  
Microfilament Bundle ◽  
Downstream Effectors ◽  
Fibroblast Adhesion ◽  
Rho Kinases ◽  
Phagocytic Uptake

The homologous mammalian rho kinases (ROCK I and II) are assumed to be functionally redundant, based largely on kinase construct overexpression. As downstream effectors of Rho GTPases, their major substrates are myosin light chain and myosin phosphatase. Both kinases are implicated in microfilament bundle assembly and smooth muscle contractility. Here, analysis of fibroblast adhesion to fibronectin revealed that although ROCK II was more abundant, its activity was always lower than ROCK I. Specific reduction of ROCK I by siRNA resulted in loss of stress fibers and focal adhesions, despite persistent ROCK II and guanine triphosphate–bound RhoA. In contrast, the microfilament cytoskeleton was enhanced by ROCK II down-regulation. Phagocytic uptake of fibronectin-coated beads was strongly down-regulated in ROCK II–depleted cells but not those lacking ROCK I. These effects originated in part from distinct lipid-binding preferences of ROCK pleckstrin homology domains. ROCK II bound phosphatidylinositol 3,4,5P3 and was sensitive to its levels, properties not shared by ROCK I. Therefore, endogenous ROCKs are distinctly regulated and in turn are involved with different myosin compartments.

scholarly journals Blockade of ROCK inhibits migration of human primary keratinocytes and malignant epithelial skin cells by regulating actomyosin contractility

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Srisathya Srinivasan ◽  
Sreya Das ◽  
Vishakha Surve ◽  
Ankita Srivastava ◽  
Sushant Kumar ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Focal Adhesions ◽  
Primary Keratinocytes ◽  
Skin Cells ◽  
Physiological Processes ◽  
Actomyosin Contractility ◽  
Malignant Skin ◽  
And Migration ◽  
Mlc Phosphorylation

AbstractActomyosin contractility, crucial for several physiological processes including migration, is controlled by the phosphorylation of myosin light chain (MLC). Rho-associated protein kinase (ROCK) and Myosin light chain kinase (MLCK) are predominant kinases that phosphorylate MLC. However, the distinct roles of these kinases in regulating actomyosin contractility and their subsequent impact on the migration of healthy and malignant skin cells is poorly understood. We observed that blockade of ROCK in healthy primary keratinocytes (HPKs) and epidermal carcinoma cell line (A-431 cells) resulted in loss of migration, contractility, focal adhesions, stress fibres, and changes in morphology due to reduction in phosphorylated MLC levels. In contrast, blockade of MLCK reduced migration, contractile dynamics, focal adhesions and phosphorylated MLC levels of HPKs alone and had no effect on A-431 cells due to the negligible MLCK expression. Using genetically modified A-431 cells expressing phosphomimetic mutant of p-MLC, we show that ROCK dependent phosphorylated MLC controls the migration, focal adhesion, stress fibre organization and the morphology of the cells. In conclusion, our data indicate that ROCK is the major kinase of MLC phosphorylation in both HPKs and A-431 cells, and regulates the contractility and migration of healthy as well as malignant skin epithelial cells.

scholarly journals The RhoA-ROCK pathway in the regulation of T and B cell responses

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 2295 ◽  
Author(s):  
Edd Ricker ◽  
Luvana Chowdhury ◽  
Woelsung Yi ◽  
Alessandra B. Pernis
Keyword(s):  
Immune Responses ◽  
Biological Processes ◽  
Dynamic Interactions ◽  
Cell Responses ◽  
Important Mediator ◽  
Downstream Effectors ◽  
Wide Range ◽  
Response To Stress ◽  
The Impact ◽  
Rho Kinases

Effective immune responses require the precise regulation of dynamic interactions between hematopoietic and non-hematopoietic cells. The Rho subfamily of GTPases, which includes RhoA, is rapidly activated downstream of a diverse array of biochemical and biomechanical signals, and is emerging as an important mediator of this cross-talk. Key downstream effectors of RhoA are the Rho kinases, or ROCKs. The ROCKs are two serine-threonine kinases that can act as global coordinators of a tissue’s response to stress and injury because of their ability to regulate a wide range of biological processes. Although the RhoA-ROCK pathway has been extensively investigated in the non-hematopoietic compartment, its role in the immune system is just now becoming appreciated. In this commentary, we provide a brief overview of recent findings that highlight the contribution of this pathway to lymphocyte development and activation, and the impact that dysregulation in the activation of RhoA and/or the ROCKs may exert on a growing list of autoimmune and lymphoproliferative disorders.

scholarly journals Constitutive p21-activated Kinase (PAK) Activation in Breast Cancer Cells as a Result of Mislocalization of PAK to Focal Adhesions

2004 ◽  
Vol 15 (6) ◽  
pp. 2965-2977 ◽  
Author(s):  
Mary R. Stofega ◽  
Luraynne C. Sanders ◽  
Elisabeth M. Gardiner ◽  
Gary M. Bokoch
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Rho Gtpases ◽  
Breast Cancer Cells ◽  
Kinase Activity ◽  
Focal Adhesions ◽  
Breast Cancer Cell Lines ◽  
Cytoskeletal Remodeling ◽  
P21 Activated Kinase ◽  
Pak Kinase

Cytoskeletal remodeling is critical for cell adhesion, spreading, and motility. p21-activated kinase (PAK), an effector molecule of the Rho GTPases Rac and Cdc42, has been implicated in cytoskeletal remodeling and cell motility. PAK kinase activity and subcellular distribution are tightly regulated by rapid and transient localized Rac and Cdc42 activation, and by interactions mediated by adapter proteins. Here, we show that endogenous PAK is constitutively activated in certain breast cancer cell lines and that this active PAK is mislocalized to atypical focal adhesions in the absence of high levels of activated Rho GTPases. PAK localization to focal adhesions in these cells is independent of PAK kinase activity, NCK binding, or GTPase binding, but requires the association of PAK with PIX. Disruption of the PAK–PIX interaction with competitive peptides displaces PAK from focal adhesions and results in a substantial reduction in PAK hyperactivity. Moreover, disruption of the PAK–PIX interaction is associated with a dramatic decrease of PIX and paxillin in focal adhesions, indicating that PAK localization to these structures via PIX is required for the maintenance of paxillin- and PIX-containing focal adhesions. Abnormal regulation of PAK localization and activity may contribute to the tumorigenic properties of certain breast cancer cells.

scholarly journals Targeting and activation of Rac1 are mediated by the exchange factor β-Pix

2006 ◽  
Vol 172 (5) ◽  
pp. 759-769 ◽  
Author(s):  
Jean Paul ten Klooster ◽  
Zahara M. Jaffer ◽  
Jonathan Chernoff ◽  
Peter L. Hordijk
Keyword(s):  
Rho Gtpases ◽  
Molecular Mechanisms ◽  
Rho Gtpase ◽  
Focal Adhesions ◽  
Exchange Factor ◽  
Downstream Signaling ◽  
Intracellular Targeting ◽  
Dynamics And Control ◽  
P21 Activated Kinase ◽  
Gtpase Activation

Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator β-Pix (p21-activated kinase [Pak]–interacting exchange factor). The interaction with β-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1–β-Pix interaction is required for Rac1 activation by β-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the β-Pix–binding kinase Pak1, we show that Pak1 regulates the Rac1–β-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor β-Pix.

scholarly journals Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics

2007 ◽  
Vol 176 (5) ◽  
pp. 667-680 ◽  
Author(s):  
Markus Schober ◽  
Srikala Raghavan ◽  
Maria Nikolova ◽  
Lisa Polak ◽  
H. Amalia Pasolli ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Focal Adhesions ◽  
Cellular Responses ◽  
Stress Fibers ◽  
Integrin Signaling ◽  
Cytoskeletal Dynamics ◽  
Skin Epidermis

In response to αβ1 integrin signaling, transducers such as focal adhesion kinase (FAK) become activated, relaying to specific machineries and triggering distinct cellular responses. By conditionally ablating Fak in skin epidermis and culturing Fak-null keratinocytes, we show that FAK is dispensable for epidermal adhesion and basement membrane assembly, both of which require αβ1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of αβ1 integrin in regulating cytoskeletal dynamics and orchestrating polarized keratinocyte migration out of epidermal explants. Fak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with robust, peripheral focal adhesions and microtubules. We find that without FAK, Src, p190RhoGAP, and PKL–PIX–PAK, localization and/or activation at focal adhesions are impaired, leading to elevated Rho activity, phosphorylation of myosin light chain kinase, and enhanced tensile stress fibers. We show that, together, these FAK-dependent activities are critical to control the turnover of focal adhesions, which is perturbed in the absence of FAK.

scholarly journals Adhesion, growth, and matrix production by fibroblasts on laminin substrates.

1983 ◽  
Vol 96 (1) ◽  
pp. 177-183 ◽  
Author(s):  
J R Couchman ◽  
M Höök ◽  
D A Rees ◽  
R Timpl
Keyword(s):  
Extracellular Matrix ◽  
Protein Synthesis ◽  
Focal Adhesions ◽  
Skin Fibroblasts ◽  
Subsequent Growth ◽  
Microfilament Bundles ◽  
Extracellular Matrix Production ◽  
Embryonic Skin ◽  
Fibroblast Adhesion ◽  
Matrix Production

Human embryonic skin fibroblasts have been shown to attach and spread on laminin substrates in the absence of protein synthesis and presence of fibronectin-depleted serum and anti-fibronectin antibodies. Rates of attachment and the type of spreading are virtually identical on fibronectin and laminin-coated substrates with the development of microfilament bundles and focal adhesions. Antibodies to laminin, but not fibronectin, will prevent or reverse fibroblast adhesion to laminin, whereas antibodies to fibronectin but not laminin will give similar results on fibronectin-coated substrates. These and other results indicate that fibroblasts possess distinct receptors for laminin and fibronectin which on contact with suitable substrates promote adhesion through interaction with common intermediates. This type of adhesion is compatible with subsequent growth and extracellular matrix production.

scholarly journals PAK Kinases Target Sortilin and Modulate Its Sorting

2019 ◽  
Vol 40 (3) ◽  
Author(s):  
Lone Tjener Pallesen ◽  
Camilla Gustafsen ◽  
Jacob Flyvholm Cramer ◽  
Steen Vang Petersen ◽  
Søren Skou Thirup ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Intracellular Transport ◽  
Intracellular Localization ◽  
Adaptor Proteins ◽  
Serine Residue ◽  
Endosome Trafficking ◽  
Downstream Effectors ◽  
Dileucine Motif ◽  
Group A ◽  
Kinase Phosphorylation

ABSTRACT The multifunctional type 1 receptor sortilin is involved in endocytosis and intracellular transport of ligands. The short intracellular domain of sortilin binds several cytoplasmic adaptor proteins (e.g., the AP-1 complex and GGA1 to -3), most of which target two well-defined motifs: a C-terminal acidic cluster dileucine motif and a YXXΦ motif in the proximal third of the domain. Both motifs contribute to endocytosis as well as Golgi-endosome trafficking of sortilin. The C-terminal acidic cluster harbors a serine residue, which is subject to phosphorylation by casein kinase. Phosphorylation of this serine residue is known to modulate adaptor binding to sortilin. Here, we show that the cytoplasmic domain of sortilin also engages Rac-p21-activated kinases 1 to 3 (PAK1-3) via a binding segment that includes a tyrosine-based motif, also encompassing a serine residue. We further demonstrate that PAK1-3 specifically phosphorylate this serine residue and that this phosphorylation alters the affinity for AP-1 binding and consequently changes the intracellular localization of sortilin as a result of modulated trafficking. Our findings suggest that trafficking of ligands bound to sortilin is in part regulated by group A PAK kinases, which are downstream effectors of Rho GTPases and are known to affect a variety of processes by remodeling the cytoskeleton and by promoting gene transcription and cell survival.

scholarly journals Hypoxic modulation of fetal vascular MLCK abundance, localization, and function

2021 ◽  
Vol 320 (1) ◽  
pp. R1-R18
Author(s):  
Dane W. Sorensen ◽  
Desirelys Carreon ◽  
James M. Williams ◽  
William J. Pearce
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Carotid Arteries ◽  
Chronic Hypoxia ◽  
Contractile Apparatus ◽  
Smooth Muscle Contractility ◽  
Heterogeneous Distribution ◽  
Regulatory Myosin Light Chain ◽  
And Function

Changes in vascular contractility are among the most important physiological effects of acute and chronic fetal hypoxia. Given the essential role of myosin light-chain kinase (MLCK) in smooth muscle contractility and its heterogeneous distribution, this study explores the hypothesis that subcellular changes in MLCK distribution contribute to hypoxic modulation of fetal carotid artery contractility. Relative to common carotid arteries from normoxic term fetal lambs (FN), carotids from fetal lambs gestated at high altitude (3,802 m) (FH) exhibited depressed contractility without changes in MLCK mRNA or protein abundance. Patterns of confocal colocalization of MLCK with α-actin and 20-kDa regulatory myosin light chain (MLC20) enabled calculation of subcellular MLCK fractions: 1) colocalized with the contractile apparatus, 2) colocalized with α-actin distant from the contractile apparatus, and 3) not colocalized with α-actin. Chronic hypoxia did not affect MLCK abundance in the contractile fraction, despite a concurrent decrease in contractility. Organ culture for 72 h under 1% O2 decreased total MLCK abundance in FN and FH carotid arteries, but decreased the contractile MLCK abundance only in FH carotid arteries. Correspondingly, culture under 1% O2 depressed contractility more in FH than FN carotid arteries. In addition, hypoxia appeared to attenuate ubiquitin-independent proteasomal degradation of MLCK, as reported for other proteins. In aggregate, these results demonstrate that the combination of chronic hypoxia followed by hypoxic culture can induce MLCK translocation among at least three subcellular fractions with possible influences on contractility, indicating that changes in MLCK distribution are a significant component of fetal vascular responses to hypoxia.

Heme oxygenase modulates oxidant-signaled airway smooth muscle contractility: role of bilirubin

2002 ◽  
Vol 283 (3) ◽  
pp. L596-L603 ◽  
Author(s):  
Abdoulaye Samb ◽  
Camille Taillé ◽  
Abdelhamid Almolki ◽  
Jérôme Mégret ◽  
James M. Staddon ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Heme Oxygenase ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Contractile Response ◽  
Redox Status ◽  
Ros Production ◽  
Smooth Muscle Contractility

Reactive oxygen species (ROS) increase the contractile response of airway smooth muscle (ASM). Heme oxygenase (HO) catabolizes heme to the powerful antioxidant bilirubin. Because HO is expressed in the airways, we investigated its effects on ASM contractility and ROS production in guinea pig trachea. HO expression was higher in the epithelium than in tracheal smooth muscle. Incubation of tracheal rings (TR) with the HO inhibitor tin protoporphyrin (SnPP IX) or the HO substrate hemin increased and decreased, respectively, ASM contractile response to carbamylcholine. The effect of hemin was reversed by SnPP and mimicked by the antioxidants superoxide dismutase (SOD) and catalase. Hemin significantly reduced the effect of carbamylcholine in rings treated with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), compared with ODQ-treated rings without hemin incubation, suggesting that the CO-guanosine 3′,5′-cyclic monophosphate pathway was not involved in the control of tracheal reactivity. SnPP and hemin increased and decreased ROS production by TR by 18 and 38%, respectively. Bilirubin (100 pM) significantly decreased TR contractility and ROS production. Hemin, bilirubin, and SOD/catalase decreased phosphorylation of the contractile protein myosin light chain, whereas SnPP significantly augmented it. These data suggest that modulation of the redox status by HO and, moreover, by bilirubin modulates ASM contractility by modulating levels of phosphorylated myosin light chain.

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