scholarly journals Targeting Focal Adhesion Assembly by Ethoxyfagaronine Prevents Lymphoblastic Cell Adhesion to Fibronectin

2012 ◽  
Vol 35 (4) ◽  
pp. 267-284 ◽  
Author(s):  
F. Ouchani ◽  
J. Devy ◽  
A. Rusciani ◽  
J. J. Helesbeux ◽  
S. Salesse ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Leukemic Cell ◽  
Kinase Assay ◽  
Β1 Integrin ◽  
A Cell ◽  
Synthetic Derivative ◽  
Cell Retraction

Background: Leukemic cell adhesion to proteins of the bone marrow microenvironment provides signals which control morphology, motility and cell survival. We described herein the ability of ethoxyfagaronine (etxfag), a soluble synthetic derivative of fagaronine, to prevent leukemic cell adhesion to fibronectin peptide (FN/V).Methods: Phosphorylation of fak and pyk2 were evaluated by immunoblotting. Labelled proteins were localized by confocal microscopy. PI 3-kinase activity was evaluated byin vitrokinase assay.Results: Subtoxic concentration of etxfag reduced L1210 cell adhesion to FN/V dependently of β1 integrin engagement. Etxfag impaired FN-dependent formation of β1 clustering without modifying β1 expression at the cell membrane. This was accompanied by a decrease of focal adhesion number, a diminution of fak and pyk2 phosphorylation at Tyr-576, Tyr-861 and Tyr-579, respectively leading to their dissociations from β1 integrin and inhibition of PI 3-kinase activity. Etxfag also induced a cell retraction accompanied by a redistribution of phosphorylated fak and pyk2 in the perinuclear region and lipid raft relocalization.Conclusion: Through its anti-adhesive potential, etxfag, combined with conventional cytotoxic drugs could be potentially designed as a new anti-leukemic drug.

scholarly journals Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases.

1995 ◽  
Vol 15 (2) ◽  
pp. 954-963 ◽  
Author(s):  
M B Calalb ◽  
T R Polte ◽  
S K Hanks
Keyword(s):  
Signal Transduction ◽  
Cell Adhesion ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Src Family Kinases ◽  
Protein Kinase Activity ◽  
Dependent Manner

Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein-tyrosine kinase implicated in integrin-mediated signal transduction pathways and in the process of oncogenic transformation by v-Src. Elevation of FAK's phosphotyrosine content, following both cell adhesion to extracellular matrix substrata and cell transformation by Rous sarcoma virus, correlates directly with an increased kinase activity. To help elucidate the role of FAK phosphorylation in signal transduction events, we used a tryptic phosphopeptide mapping approach to identify tyrosine sites of phosphorylation responsive to both cell adhesion and Src transformation. We have identified four tyrosines, 397, 407, 576, and 577, which are phosphorylated in mouse BALB/3T3 fibroblasts in an adhesion-dependent manner. Tyrosine 397 has been previously recognized as the major site of FAK autophosphorylation. Phosphorylation of tyrosines 407, 576, and 577, which are previously unrecognized sites, is significantly elevated in the presence of c-Src in vitro and v-Src in vivo. Tyrosines 576 and 577 lie within catalytic subdomain VIII--a region recognized as a target for phosphorylation-mediated regulation of protein kinase activity. We found that maximal kinase activity of FAK immune complexes requires phosphorylation of both tyrosines 576 and 577. Our results indicate that phosphorylation of FAK by Src (or other Src family kinases) is an important step in the formation of an active signaling complex.

Neuromuscular target recognition by a homophilic interaction of connectin cell adhesion molecules in Drosophila

Development ◽  
1997 ◽  
Vol 124 (8) ◽  
pp. 1433-1441 ◽  
Author(s):  
A. Nose ◽  
T. Umeda ◽  
M. Takeichi
Keyword(s):  
Cell Adhesion ◽  
Synapse Formation ◽  
Target Recognition ◽  
Surface Protein ◽  
Transgenic Lines ◽  
A Cell ◽  
Neuromuscular Connectivity ◽  
Recognition Molecule

Drosophila Connectin (CON) is a cell surface protein of the leucine-rich repeat family. During the formation of neuromuscular connectivity, CON is expressed on the surface of a subset of embryonic muscles and on the growth cones and axons of the motoneurons that innervate these muscles, including primarily SNa motoneurons and their synaptic targets (lateral muscles). In vitro, CON can mediate homophilic cell adhesion. In this study, we generated transgenic lines that ectopically expressed CON on all muscles. In the transformant embryos and larvae, SNa motoneurons often inappropriately innervated a neighboring non-target muscle (muscle 12) that ectopically expressed CON. Furthermore, the ectopic synapse formation was dependent on the endogenous CON expression on the SNa motoneurons. These results show that CON can function as an attractive and homophilic target recognition molecule in vivo.

scholarly journals Phosphorylation of IkappaBalpha in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability.

1996 ◽  
Vol 16 (4) ◽  
pp. 1401-1409 ◽  
Author(s):  
R Lin ◽  
P Beauparlant ◽  
C Makris ◽  
S Meloche ◽  
J Hiscott
Keyword(s):  
Triple Point ◽  
Cell Activation ◽  
Kinase Activity ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Kinase Assay ◽  
Intrinsic Stability ◽  
Nf Kappab ◽  
Constitutive Phosphorylation

The NF-kappaB/Rel transcription factors participate in the activation of immune system regulatory genes and viral early genes including the human immunodeficiency virus type 1 long terminal repeat. NF-kappaB/Rel proteins are coupled to inhibitory molecules, collectively termed IkappaB, which are responsible for cytoplasmic retention of NF-kappaB. Cell activation leads to the phosphorylation and degradation of IkappaBalpha, permitting NG-kappaB/Rel translocation to the nucleus and target gene activation. To further characterize the signaling events that contribute to IkappaBalpha phosphorylation, a kinase activity was isolated from Jurkat T cells that specifically interacted with IkappaBalpha in an affinity chromatography step and phosphorylated IkappaBalpha with high specificity in vitro. By using an in-gel kinase assay with recombinant IkappaBalpha as substrate, two forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria and immunological cross-reactivity identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Deletion mutants of IkappaBalpha delta1 to delta4) localized phosphorylation to the C-terminal PEST domain of IkappaBalpha. Point mutation of residues T-291, S-283, and T-299 dramatically reduced phosphorylation of IkappaBalpha by the kinase in vitro. NIH-3T3 cells that stably expressed wild-type IkappaBalpha (wtIkappaB), double-point-mutated IkappaBalpha (T291A, S283A), or triple-point-mutated IkappaBalpha (T291A, S283A, T299A) under the control of the tetracycline-responsive promoter were generated. Constitutive phosphorylation of the triple point mutant was eliminated in vivo, although tumor necrosis factor-inducible IkappaBalpha degradation was unaffected. In cell lines and in transiently transfected cells, mutation of the CKII sites in IkappaBalpha resulted in a protein with increased intrinsic stability. Together with results demonstrating a role for N-terminal sites in inducer-mediated phosphorylation and degradation of IkappaBalpha, these studies indicate that CKII sites in the C-terminal PEST domain are important for constitutive phosphorylation and intrinsic stability of IkappaBalpha.

CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2633-2639 ◽  
Author(s):  
Atsushi Oda ◽  
Hans D. Ochs ◽  
Laurence A. Lasky ◽  
Susan Spencer ◽  
Katsutoshi Ozaki ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Phosphorylation ◽  
Sh3 Domain ◽  
Cytoskeletal Proteins ◽  
Kinase Assay ◽  
Sh3 Domains ◽  
Wiskott Aldrich Syndrome ◽  
Dynamic Cell ◽  
Syndrome Protein

Abstract Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia are caused by mutations of the WAS protein (WASP) gene. WASP may be involved in the regulation of podosome, an actin-rich dynamic cell adhesion structure formed by various types of cells. The molecular links between WASP and podosomes or other cell adhesion structures are unknown. Platelets express an SH2-SH3 adapter molecule, CrkL, that can directly associate with paxillin, which is localized in podosomes. The hypothesis that CrkL binds to WASP was, therefore, tested. Results from coprecipitation experiments using anti-CrkL and GST-fusion proteins suggest that CrkL binds to WASP through its SH3 domain and that the binding was not affected by WASP tyrosine phosphorylation. The binding of GST-fusion SH3 domain of PSTPIP1 in vitro was also not affected by WASP tyrosine phosphorylation, suggesting that the binding of the SH3 domains to WASP is not inhibited by tyrosine phosphorylation of WASP. Anti-CrkL also coprecipitates a 72-kd protein, which was identified as syk tyrosine kinase, critical for collagen induced-platelet activation. CrkL immunoprecipitates contain kinase-active syk, as evidenced by an in vitro kinase assay. Coprecipitation experiments using GST-fusion CrkL proteins suggest that both SH2 and SH3 domains of CrkL are involved in the binding of CrkL to syk. WASP, CrkL, syk, and paxillin-like Hic-5 incorporated to platelet cytoskeleton after platelet aggregation. Thus, CrkL is a novel molecular adapter for WASP and syk and may potentially transfer these molecules to the cytoskeleton through association with cytoskeletal proteins such as Hic-5.

Arabidopsis MLK3, a Plant-specific Casein Kinase 1, Negatively Regulated Flowering and Phosphorylated Histone H3 in vivo

2019 ◽  
Author(s):  
Zhen Wang ◽  
Junmei Kang ◽  
Shangang Jia ◽  
Tiejun Zhang ◽  
Zhihai Wu ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Histone H3 ◽  
Casein Kinase ◽  
Kinase Assay ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
Altered Expression ◽  
Casein Kinase 1 ◽  
Threonine Kinase

Abstract Background: Casein kinase 1 (CK1) family members are highly conserved serine/threonine kinase present in most eukaryotes with multiple biological functions. Arabidopsis MUT9-like kinases ( MLKs ) belong to a clade CK1 specific to the plant kingdom and have been implicated collectively in modulating flowering related processes. Three of the four MLKs ( MLK1/2/4 ) have been characterized, however, little is known about MLK3 , the most divergent MLKs. Results: We demonstrated that compared with wild type, mlk3 , a truncated MLK3 , flowered slightly early under long day conditions and ectopic expression of MLK3 rescued the morphological defects of mlk3 , indicating that MLK3 negatively regulates flowering. GA 3 application accelerated flowering of both wild type and mlk3 , suggesting that mlk3 had normal GA response. The recombinant MLK3-GFP was localized in the nucleus exclusively. In vitro kinase assay revealed that the nuclear protein MLK3 phosphorylated histone 3 at threonine 3 (H3T3ph). Mutation of a conserved catalytic residue (Lysine 175) abolished the kinase activity and resulted in failure to complement the early flowering phenotype of mlk3 . Interestingly, the global level of H3T3 phosphorylation in mlk3 did not differ significantly from wild type, suggesting the redundant roles of MLKs in flowering regulation. The transcriptomic analysis demonstrated that 425 genes significantly altered expression level in mlk3 relative to wild type. The mlk3 mlk4 double mutant generated by crossing mlk3 with mlk4 , a loss-of-function mutant of MLK4 showing late flowering, flowered between the two parental lines, suggesting that MLK3 played an antagonistic role to MLK4 in plant transition to flowering. Conclusions: A serine/threonine kinase encoding gene MLK3 is a casein kinase 1 specific to the plant species and represses flowering slightly. MLK3 located in nucleus catalyzes the phosphorylation of histone H3 at threonine 3 in vitro and an intact lysine residue (K175) is indispensible for the kinase activity. This study sheds new light on the delicate control of flowering by the plant-specific CK1 in Arabidopsis.

scholarly journals Polo-Like Kinase 1 as a Target for Human Cytomegalovirus pp65 Lower Matrix Protein

1999 ◽  
Vol 73 (2) ◽  
pp. 1468-1478 ◽  
Author(s):  
A. Gallina ◽  
L. Simoncini ◽  
S. Garbelli ◽  
E. Percivalle ◽  
G. Pedrali-Noy ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Matrix Protein ◽  
Kinase Activity ◽  
Major Constituent ◽  
Early Protein ◽  
Vitro Binding ◽  
A Cell ◽  
Bona Fide ◽  
Hydrophilic Region

ABSTRACT Human cytomegalovirus (HCMV) pp65 protein is the major constituent of viral dense bodies but is dispensable for viral growth in vitro. pp65 copurifies with a S/T kinase activity and has been implicated in phosphorylation of HCMV IE1 immediate-early protein and its escape from major histocompatibility complex 1 presentation. Furthermore, the presence of pp65 correlates with a virion-associated kinase activity. To clarify the role of pp65, yeast two-hybrid system (THS) screening was performed to identify pp65 cellular partners. A total of 18 out of 48 yeast clones harboring cDNAs for putative pp65 binding proteins encoded the Polo-like kinase 1 (Plk1) C-terminal domain. Plk1 behaved as a bona fide pp65 partner in THS control crosses, and the interaction was confirmed by in vitro binding experiments. Endogenous Plk1 was coimmunoprecipitated with pp65 from transiently transfected COS7 cells. In infected fibroblasts, Plk1 was coimmunoprecipitated with pp65 at late infection stages. Furthermore, Plk1 was detected within wild-type HCMV particles but not within the particles of a pp65-negative mutant (RVAd65). The hydrophilic region of pp65 was phosphorylated in vitro by Plk1. These results suggest that one function of pp65 may be to capture a cell kinase, perhaps in order to alter its activity, nucleotide preference, substrate specificity, or subcellular localization to the advantage of HCMV.

scholarly journals Cell adhesion molecule IGPR-1 activates AMPK connecting cell adhesion to autophagy

2020 ◽  
Vol 295 (49) ◽  
pp. 16691-16699
Author(s):  
Razie Amraei ◽  
Tooba Alwani ◽  
Rachel Xi-Yeen Ho ◽  
Zahra Aryan ◽  
Shawn Wang ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Cellular Assays ◽  
Iκb Kinase ◽  
Promising Therapeutic Target ◽  
A Cell ◽  
Subsequent Activation

Autophagy plays critical roles in the maintenance of endothelial cells in response to cellular stress caused by blood flow. There is growing evidence that both cell adhesion and cell detachment can modulate autophagy, but the mechanisms responsible for this regulation remain unclear. Immunoglobulin and proline-rich receptor-1 (IGPR-1) is a cell adhesion molecule that regulates angiogenesis and endothelial barrier function. In this study, using various biochemical and cellular assays, we demonstrate that IGPR-1 is activated by autophagy-inducing stimuli, such as amino acid starvation, nutrient deprivation, rapamycin, and lipopolysaccharide. Manipulating the IκB kinase β activity coupled with in vivo and in vitro kinase assays demonstrated that IκB kinase β is a key serine/threonine kinase activated by autophagy stimuli and that it catalyzes phosphorylation of IGPR-1 at Ser220. The subsequent activation of IGPR-1, in turn, stimulates phosphorylation of AMP-activated protein kinase, which leads to phosphorylation of the major pro-autophagy proteins ULK1 and Beclin-1 (BECN1), increased LC3-II levels, and accumulation of LC3 punctum. Thus, our data demonstrate that IGPR-1 is activated by autophagy-inducing stimuli and in response regulates autophagy, connecting cell adhesion to autophagy. These findings may have important significance for autophagy-driven pathologies such cardiovascular diseases and cancer and suggest that IGPR-1 may serve as a promising therapeutic target.

A rapid, multiplexed kinase activity assay using 8-plex iTRAQ labeling, SPE, and MALDI-TOF/TOF MS

The Analyst ◽  
2020 ◽  
Vol 145 (3) ◽  
pp. 992-1000
Author(s):  
Yu-Ching Liu ◽  
Fuu-Jen Tsai ◽  
Chao-Jung Chen
Keyword(s):  
Kinase Activity ◽  
Kinase Assay ◽  
Activity Assay ◽  
Peptide Substrates ◽  
Itraq Labeling ◽  
Cell Lysates ◽  
Maldi Tof ◽  
Kinase Activity Assay ◽  
Tof Ms

Synthesized peptide substrates with iTRAQ labeling has been used for in vitro kinase assay using cell lysates and detected by MALDI TOF/TOF MS.

scholarly journals The Human Papillomavirus E6 Oncogene Represses a Cell Adhesion Pathway and Disrupts Focal Adhesion through Degradation of TAp63β upon Transformation

2011 ◽  
Vol 7 (9) ◽  
pp. e1002256 ◽  
Author(s):  
Youcef Ben Khalifa ◽  
Sébastien Teissier ◽  
Meng-Kwang Marcus Tan ◽  
Quang Tien Phan ◽  
Mathieu Daynac ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Human Papillomavirus ◽  
Focal Adhesion ◽  
A Cell
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