scholarly journals Proteomics analysis identifies PEA-15 as an endosomal phosphoprotein that regulates α5β1 integrin endocytosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maisel J. Caliva ◽  
Won Seok Yang ◽  
Shirley Young-Robbins ◽  
Ming Zhou ◽  
Hana Yoon ◽  
...  
Keyword(s):  
Cell Motility ◽  
Cellular Migration ◽  
Endosomal Trafficking ◽  
Matrix Assembly ◽  
Transmembrane Receptors ◽  
Surface Receptors ◽  
Proximity Ligation ◽  
Assembly Cell ◽  
Proteomics Approach ◽  
Α5β1 Integrin

AbstractEndosomal trafficking of cell surface receptors is essential to their function. Integrins are transmembrane receptors that integrate adhesion to the extracellular matrix with engagement of the cytoskeleton. Ligated integrins mediate diverse signals that regulate matrix assembly, cell survival, cell morphology, and cell motility. Endosomal trafficking of integrins modulates these signals and contributes to cell motility and is required for cancer cell invasion. The phosphoprotein PEA-15 modulates integrin activation and ERK MAP Kinase signaling. To elucidate novel PEA-15 functions we utilized an unbiased proteomics approach. We identified several binding partners for PEA-15 in the endosome including clathrin and AP-2 as well as integrin β1 and other focal adhesion complex proteins. We confirmed these interactions using proximity ligation analysis, immunofluorescence imaging, pull-down and co-immunoprecipitation. We further found that PEA-15 is enriched in endosomes and was required for efficient endosomal internalization of α5β1 integrin and cellular migration. Importantly, PEA-15 promotion of migration was dependent on PEA-15 phosphorylation at serines 104 and 116. These data support a novel endosomal role for PEA-15 in control of endosomal trafficking of integrins through an association with the β1 integrin and clathrin complexes, and thereby regulation of cell motility.

scholarly journals Activation of Distinct α5β1-mediated Signaling Pathways by Fibronectin's Cell Adhesion and Matrix Assembly Domains

1998 ◽  
Vol 141 (1) ◽  
pp. 241-253 ◽  
Author(s):  
Denise C. Hocking ◽  
Jane Sottile ◽  
Paula J. McKeown-Longo
Keyword(s):  
Cell Adhesion ◽  
Amino Terminus ◽  
Type I ◽  
New Paradigm ◽  
Matrix Assembly ◽  
Rgd Peptides ◽  
Surface Receptors ◽  
Amino Terminal ◽  
Dynamic Cell ◽  
Α5β1 Integrin

The interaction of cells with fibronectin generates a series of complex signaling events that serve to regulate several aspects of cell behavior, including growth, differentiation, adhesion, and motility. The formation of a fibronectin matrix is a dynamic, cell-mediated process that involves both ligation of the α5β1 integrin with the Arg-Gly-Asp (RGD) sequence in fibronectin and binding of the amino terminus of fibronectin to cell surface receptors, termed “matrix assembly sites,” which mediate the assembly of soluble fibronectin into insoluble fibrils. Our data demonstrate that the amino-terminal type I repeats of fibronectin bind to the α5β1 integrin and support cell adhesion. Furthermore, the amino terminus of fibronectin modulates actin assembly, focal contact formation, tyrosine kinase activity, and cell migration. Amino-terminal fibronectin fragments and RGD peptides were able to cross-compete for binding to the α5β1 integrin, suggesting that these two domains of fibronectin cannot bind to the α5β1 integrin simultaneously. Cell adhesion to the amino-terminal domain of fibronectin was enhanced by cytochalasin D, suggesting that the ligand specificity of the α5β1 integrin is regulated by the cytoskeleton. These data suggest a new paradigm for integrin-mediated signaling, where distinct regions within one ligand can modulate outside-in signaling through the same integrin.

scholarly journals Modulation of Cellular Migration and Survival by c-Myc through the Downregulation of Urokinase (uPA) and uPA Receptor

2010 ◽  
Vol 30 (7) ◽  
pp. 1838-1851 ◽  
Author(s):  
Daniela Alfano ◽  
Giuseppina Votta ◽  
Almut Schulze ◽  
Julian Downward ◽  
Mario Caputi ◽  
...  
Keyword(s):  
Cell Motility ◽  
Cellular Transformation ◽  
Tumor Formation ◽  
Cellular Migration ◽  
Epithelial Cell Line ◽  
Cellular Motility ◽  
Upa Receptor ◽  
Upar Expression ◽  
Repressive Effect ◽  
Cell Invasiveness

ABSTRACT It has been proposed that c-Myc proapoptotic activity accounts for most of its restraint of tumor formation. We established a telomerase-immortalized human epithelial cell line expressing an activatable c-Myc protein. We found that c-Myc activation induces, in addition to increased sensitivity to apoptosis, reductions in cell motility and invasiveness. Transcriptome analysis revealed that urokinase (uPA) and uPA receptor (uPAR) were strongly downregulated by c-Myc. Evidence is provided that the repression of uPA and uPAR may account for most of the antimigratory and proapoptotic activities of c-Myc. c-Myc is known to cooperate with Ras in cellular transformation. We therefore investigated if this cooperation could converge in the control of uPA/uPAR expression. We found that Ras is able to block the effects of c-Myc activation on apoptosis and cellular motility but not on cell invasiveness. Accordingly, the activation of c-Myc in the context of Ras expression had only minor influence on uPAR expression but still had a profound repressive effect on uPA expression. Thus, the differential regulation of uPA and uPAR by c-Myc and Ras correlates with the effects of these two oncoproteins on cell motility, invasiveness, and survival. In conclusion, we have discovered a novel link between c-Myc and uPA/uPAR. We propose that reductions of cell motility and invasiveness could contribute to the inhibition of tumorigenesis by c-Myc and that the regulation of uPA and uPAR expression may be a component of the ability of c-Myc to reduce motility and invasiveness.

scholarly journals c-Cbl and Cbl-b ubiquitin ligases: substrate diversity and the negative regulation of signalling responses

2005 ◽  
Vol 391 (2) ◽  
pp. 153-166 ◽  
Author(s):  
Christine B. F. Thien ◽  
Wallace Y. Langdon
Keyword(s):  
Tyrosine Kinases ◽  
Ubiquitin Ligases ◽  
Adaptor Proteins ◽  
Kinase Domain ◽  
Protein Tyrosine Kinases ◽  
Tyrosine Kinase Domain ◽  
Transmembrane Receptors ◽  
Surface Receptors ◽  
Cbl Proteins ◽  
Initial Line

The activation of signalling pathways by ligand engagement with transmembrane receptors is responsible for determining many aspects of cellular function and fate. While these outcomes are initially determined by the nature of the ligand and its receptor, it is also essential that intracellular enzymes, adaptor proteins and transcription factors are correctly assembled to convey the intended response. In recent years, it has become evident that proteins that regulate the amplitude and duration of these signalling responses are also critical in determining the function and fate of cells. Of these, the Cbl family of E3 ubiquitin ligases and adaptor proteins has emerged as key negative regulators of signals from many types of cell-surface receptors. The array of receptors and downstream signalling proteins that are regulated by Cbl proteins is diverse; however, in most cases, the receptors have a common link in that they either possess a tyrosine kinase domain or they form associations with cytoplasmic PTKs (protein tyrosine kinases). Thus Cbl proteins become involved in signalling responses at a time when PTKs are first activated and therefore provide an initial line of defence to ensure that signalling responses proceed at the desired intensity and duration.

Relative quantification of protein expression in metastatic renal cell carcinoma using iTRAQ LC-MS/MS analysis reveals galectin-1 as a potential prognostic marker and therapeutic target.

2013 ◽  
Vol 31 (6_suppl) ◽  
pp. 412-412
Author(s):  
Nicole M.A. White ◽  
Olena Masui ◽  
Leroi DeSouza ◽  
Olga Krakovska ◽  
Ajay Matta ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Overall Survival ◽  
Protein Expression ◽  
Western Blot ◽  
Cell Motility ◽  
Cell Carcinoma ◽  
Therapeutic Target ◽  
Renal Cell ◽  
Cellular Migration ◽  
Metastatic Rcc

412 Background: Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant cancers. Identification of proteins involved in tumor progression will help gain a better understanding of the disease and will form the basis for the identification of novel therapeutic targets. Methods: Using six fresh-frozen primary and six unmatched metastatic RCC tumors, we used iTRAQ labeling and LC-MS/MS analysis to identify proteins differentially expressed in metastatic versus primary RCC. We verified protein expression by western blot and immunohistochemical analyses and the measured the effect of dysregulated protein expression on biological processes with RCC cell line models. Results: After analysis, we identified 29 proteins differentially expressed in metastatic versus primary RCC. We verified expressions of profilin-1, 14-3-3 zeta/delta, and galectin-1 (Gal-1) on two independent tissue sets by western blot (10 primary and 10 metastatic RCC tissues) and immunohistochemistry (22 primary and 23 metastatic tissues). Overexpression of Gal-1 in CAKI-1 cells lead to decreased actin, increased vimentin expression, and increased cellular migration. Additionally, when Gal-1 was decreased via siRNA, cells showed decreased cellular migration. Protein array analysis showed expression of cell motility-related proteins HSP27, JNK, and RSK, were altered after siRNA transfection. We also showed that Gal-1 expression was increased in response to HIF-1alpha. Furthermore, we analyzed the expression of Gal-1 mRNA in 404 RCC patients using the Cancer Genome Anatomy Project, and found that patients who had higher Gal-1 expression in the primary RCC had significantly decreased overall survival (41 vs. 78 months; p < 0.01). Conclusions: Gal-1 is increased in metastatic RCC and can effect cell migration by targeting proteins involved in cell motility. This may be a downstream effect of HIF-1α dysregulation. Decreased Gal-1 significantly decreased cellular migration suggesting Gal-1 may serve as a potential therapeutic target. Additionally, we showed that increased Gal-1 expression was associated with decreased overall survival.

Fibronectin Distribution in Human Bone Marrow Stroma: Matrix Assembly and Tumor Cell Adhesion via α5β1 Integrin

1997 ◽  
Vol 230 (1) ◽  
pp. 111-120 ◽  
Author(s):  
D. Van der Velde-Zimmermann ◽  
M.A.M. Verdaasdonk ◽  
L.H.P.M. Rademakers ◽  
R.A. De Weger ◽  
J.G. Van den Tweel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
Tumor Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Bone Marrow Stroma ◽  
Matrix Assembly ◽  
Tumor Cell Adhesion ◽  
Marrow Stroma ◽  
Α5β1 Integrin

scholarly journals PAI-1: An Integrator of Cell Signaling and Migration

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Ralf-Peter Czekay ◽  
Cynthia E. Wilkins-Port ◽  
Stephen P. Higgins ◽  
Jennifer Freytag ◽  
Jessica M. Overstreet ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Plasminogen Activator Inhibitor ◽  
Cellular Migration ◽  
Surface Binding ◽  
Surface Receptors ◽  
Activator Inhibitor Type ◽  
Pai 1 ◽  
Migratory Phenotype

Cellular migration, over simple surfaces or through complex stromal barriers, requires coordination between detachment/re-adhesion cycles, involving structural components of the extracellular matrix and their surface-binding elements (integrins), and the precise regulation of the pericellular proteolytic microenvironment. It is now apparent that several proteases and protease inhibitors, most notably urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1), also interact with several cell surface receptors transducing intracellular signals that significantly affect both motile and proliferative programs. These events appear distinct from the original function of uPA/PAI-1 as modulators of the plasmin-based proteolytic cascade. The multifaceted interactions of PAI-1 with specific matrix components (i.e., vitronectin), the low-density lipoprotein receptor-related protein-1 (LRP1), and the uPA/uPA receptor complex have dramatic consequences on the migratory phenotype and may underlie the pathophysiologic sequalae of PAI-1 deficiency and overexpression. This paper focuses on the increasingly intricate role of PAI-1 as a major mechanistic determinant of the cellular migratory phenotype.

scholarly journals TLK1-MK5 axis drives prostate cancer cell motility and pathologic features of aggressiveness

2021 ◽  
Author(s):  
Md Imtiaz Khalil ◽  
Vibha Singh ◽  
Judy King ◽  
Arrigo De Benedetti
Keyword(s):  
Prostate Cancer ◽  
Wound Healing ◽  
Cell Motility ◽  
Cell Lines ◽  
Cultured Cells ◽  
Cellular Migration ◽  
Dependent Manner ◽  
Lncap Cells ◽  
Pathologic Features ◽  
Pharmacologic Inhibition

Abstract Background: Majority of prostate cancer (PCa) related fatalities occur due to metastasis of cancer cells to adjacent and distal organs. We identified the novel interaction between two kinases (TLK1-MK5) that in part may initiate a signaling cascade promoting PCa metastasis. In PCa, TLK1-MK5 signaling might be crucial as androgen deprivation therapy leads to increased expression of TLK1 and compensatory activation of MK5 in metastatic castration-resistant prostate cancer patients. Methods: We performed scratch wound repair and 3D chemotactic migration assays to determine the motility rates of different TLK1 and MK5 perturbed cells. Co-IP, His, and GST pull down, in vitro kinase (IVK) assays and mass spectrometry (MS) were conducted to determine TLK1-MK5 interaction and phosphorylation. Western blotting (WB), immunohistochemistry (IHC) and bioinformatic analysis were used to examine TLK1 and pMK5 levels in PCa cell lines, mice prostate tumors and PCa tissue microarray (TMA). Results: Both genetic depletion and pharmacologic inhibition of TLK1 and MK5 can significantly reduce wound healing rate in MEF and LNCaP cells. However, TLK1 overexpression alone in the MK5 −/− MEF cells did not increase the wound healing which suggested that TLK1 cannot enhance cellular migration in absence of MK5. Our reciprocal co-IPs, His- and GST pull down assays confirmed TLK1-MK5 interaction in cultured cells. Incubation of purified recombinant TLK1B and MK5 increases the phosphorylation of MK5 and its kinase activity. MS analysis identified three unique phosphorylation sites in MK5 (S160, S354, S386) by TLK1B. While our WB detected substantial amount of pMK5 S354 and TLK1 in all major PCa cell lines, anti-androgen treatment increased pMK5 S354 level in a dose-dependent manner and pharmacologic inhibition of TLK1 reduces pMK5 S354 level in LNCaP cells. IHC staining of TRAMP mice prostate tissues also exhibited increased pMK5 S354 level in aggressive tumor compared to benign regions. Finally, IHC analysis of PCa TMA indicated a correlation between elevated pMK5 S354 level and generally higher Gleason scores as well as nodal metastatic status of the tumors. Conclusion: Our data support that TLK1-MK5 signaling is functionally involved in driving PCa cell motility and clinical aggressiveness, hence, disruption of this axis may inhibit the metastasis of PCa.

scholarly journals The tumor suppressor p53 promotes carcinoma invasion and collective cellular migration

2018 ◽  
Author(s):  
Shijie He ◽  
Christopher V. Carman ◽  
Jung Hyun Lee ◽  
Bo Lan ◽  
Stephan Koehler ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Colorectal Carcinoma ◽  
Cell Motility ◽  
Single Cell ◽  
Cell Layer ◽  
Carcinoma Cells ◽  
Cellular Migration ◽  
Loss Of Function ◽  
Tumor Suppressor P53 ◽  
Increase Cell Motility

SummaryLoss of function of the tumor suppressor p53 is generally thought to increase cell motility and invasiveness. Using 2-D confluent and 3-D spheroidal cell motility assays with bladder carcinoma cells and colorectal carcinoma cells, we report, to the contrary, that loss of p53 can decrease cell motility and invasion.AbstractFor migration of the single cell studied in isolation, loss of function of the tumor suppressor p53 is thought to increase cell motility. Here by contrast we used the 2-D confluent cell layer and the 3-D multicellular spheroid to investigate how p53 impacts dissemination and invasion of cellular collectives. We used two human carcinoma cell lines, the bladder carcinoma EJ and the colorectal carcinoma HCT116. We began by replicating single cell invasion in the traditional Boyden chamber assay, and found that the number of invading cells increased with loss of p53, as expected. In the confluent 2-D cell layer, however, for both EJ and HCT, speeds and effective diffusion coefficients for the p53 null types compared to their p53 expressing counterparts were significantly smaller. Compared to p53 expressers, p53 null cells exhibited more organized cortical actin rings together with reduced front-rear cell polarity. Furthermore, loss of p53 caused cells to exert smaller traction forces upon their substrates, and reduced formation of cryptic lamellipodia. In a 3-D collagen matrix, p53 consistently promoted invasion of the multicellular spheroids into surrounding matrix. Together, these results show that p53 expression in these carcinoma model systems increases collective cellular migration and invasion. As such, these studies point to paradoxical contributions of p53 in single cell versus collective cellular migration.

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