Actin depolymerization-induced tyrosine phosphorylation of cortactin: the role of Fer kinase

The F-actin-binding protein cortactin is an important regulator of cytoskeletal dynamics, and a prominent target of various tyrosine kinases. Tyrosine phosphorylation of cortactin has been suggested to reduce its F-actin cross-linking capability. In the present study, we investigated whether a reciprocal relationship exists, i.e. whether the polymerization state of actin impacts on the cortactin tyrosine phosphorylation. Actin depolymerization by LB (latrunculin B) induced robust phosphorylation of C-terminal tyrosine residues of cortactin. In contrast, F-actin stabilization by jasplakinolide, which redistributed cortactin to F-actin-containing patches, prevented cortactin phosphorylation triggered by hypertonic stress or LB. Using cell lines deficient in candidate tyrosine kinases, we found that the F-actin depolymerization-induced cortactin phosphorylation was mediated by the Fyn/Fer kinase pathway, independent of Src and c-Abl. LB caused modest Fer activation and strongly facilitated the association between Fer and cortactin. Interestingly, the F-actin-binding region within the cortactin N-terminus was essential for the efficient phosphorylation of C-terminal tyrosine residues. Investigating the structural requirements for the Fer–cortactin association, we found that (i) phosphorylation-incompetent cortactin still bound to Fer; (ii) the isolated N-terminus associated with Fer; and (iii) the C-terminus alone was insufficient for binding. Thus the cortactin N-terminus participates in the Fer–cortactin interaction, which cannot be fully due to the binding of the Fer Src homology 2 domain to C-terminal tyrosine residues of cortactin. Taken together, F-actin stabilization prevents cortactin tyrosine phosphorylation, whereas depolymerization promotes it. Depolymerization-induced phosphorylation is mediated by Fer, and requires the actin-binding domain of cortactin. These results define a novel F-actin-dependent pathway that may serve as a feedback mechanism during cytoskeleton remodelling.

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Cortactin Tyrosine Phosphorylation Requires Rac1 Activity and Association with the Cortical Actin Cytoskeleton

Molecular Biology of the Cell ◽

10.1091/mbc.e02-11-0753 ◽

2003 ◽

Vol 14 (8) ◽

pp. 3216-3229 ◽

Cited By ~ 129

Author(s):

Julie A. Head ◽

Dongyan Jiang ◽

Min Li ◽

Lynda J. Zorn ◽

Erik M. Schaefer ◽

...

Keyword(s):

Cell Motility ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Growth Factor Receptor ◽

Actin Dynamics ◽

Actin Binding ◽

Cell Periphery ◽

Carboxy Terminus ◽

Cortical Actin ◽

Cortactin Tyrosine Phosphorylation

Cortactin is an F-actin binding protein that activates actin-related protein 2/3 complex and is localized within lamellipodia. Cortactin is a substrate for Src and other protein tyrosine kinases involved in cell motility, where its phosphorylation on tyrosines 421, 466, and 482 in the carboxy terminus is required for cell movement and metastasis. In spite of the importance of cortactin tyrosine phosphorylation in cell motility, little is known regarding the structural, spatial, or signaling requirements regulating cortactin tyrosine phosphorylation. Herein, we report that phosphorylation of cortactin tyrosine residues in the carboxy terminus requires the aminoterminal domain and Rac1-mediated localization to the cell periphery. Phosphorylation-specific antibodies directed against tyrosine 421 and 466 were produced to study the regulation and localization of tyrosine phosphorylated cortactin. Phosphorylation of cortactin tyrosine 421 and 466 was elevated in response to Src, epidermal growth factor receptor and Rac1 activation, and tyrosine 421 phosphorylated cortactin localized with F-actin in lamellipodia and podosomes. Cortactin tyrosine phosphorylation is progressive, with tyrosine 421 phosphorylation required for phosphorylation of tyrosine 466. These results indicate that cortactin tyrosine phosphorylation requires Rac1-induced cortactin targeting to cortical actin networks, where it is tyrosine phosphorylated in hierarchical manner that is closely coordinated with its ability to regulate actin dynamics.

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Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50csk kinase creates a binding site for p56lck tyrosine kinase and activates the phosphatase.

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.1308 ◽

1994 ◽

Vol 14 (2) ◽

pp. 1308-1321 ◽

Cited By ~ 95

Author(s):

M Autero ◽

J Saharinen ◽

T Pessa-Morikawa ◽

M Soula-Rothhut ◽

C Oetken ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Lymphocyte Activation ◽

Sh2 Domain ◽

Protein Tyrosine Kinases ◽

Phosphotyrosine Phosphatase ◽

Intact Cells ◽

Tyrosine Residues ◽

Family Protein ◽

Ptpase Activity

Src family protein tyrosine kinases (PTKs) play an essential role in antigen receptor-initiated lymphocyte activation. Their activity is largely regulated by a negative regulatory tyrosine which is a substrate for the activating action of the CD45 phosphotyrosine phosphatase (PTPase) or, conversely, the suppressing action of the cytosolic p50csk PTK. Here we report that CD45 was phosphorylated by p50csk on two tyrosine residues, one of them identified as Tyr-1193. This residue was not phosphorylated by T-cell PTKs p56lck and p59fyn. Tyr-1193 was phosphorylated in intact T cells, and phosphorylation increased upon treatment with PTPase inhibitors, indicating that this tyrosine is a target for a constitutively active PTK. Cotransfection of CD45 and csk into COS-1 cells caused tyrosine phosphorylation of CD45 in the intact cells. Tyrosine-phosphorylated CD45 bound p56lck through the SH2 domain of the kinase. Finally, p50csk-mediated phosphorylation of CD45 caused a severalfold increase in its PTPase activity. Our results show that direct tyrosine phosphorylation of CD45 can affect its activity and association with Src family PTKs and that this phosphorylation could be mediated by p50csk. If this is also true in the intact cells, it adds a new dimension to the physiological function of p50csk in T lymphocytes.

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The Role of Protein Tyrosine Phosphatases in Inflammasome Activation

International Journal of Molecular Sciences ◽

10.3390/ijms21155481 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5481

Author(s):

Marianne R. Spalinger ◽

Marlene Schwarzfischer ◽

Michael Scharl

Keyword(s):

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Protein Complexes ◽

Protein Tyrosine Phosphatases ◽

Inflammasome Activation ◽

Tyrosine Phosphatases ◽

Tyrosine Residues ◽

Inflammasome Activity ◽

Activation Status

Inflammasomes are multi-protein complexes that mediate the activation and secretion of the inflammatory cytokines IL-1β and IL-18. More than half a decade ago, it has been shown that the inflammasome adaptor molecule, ASC requires tyrosine phosphorylation to allow effective inflammasome assembly and sustained IL-1β/IL-18 release. This finding provided evidence that the tyrosine phosphorylation status of inflammasome components affects inflammasome assembly and that inflammasomes are subjected to regulation via kinases and phosphatases. In the subsequent years, it was reported that activation of the inflammasome receptor molecule, NLRP3, is modulated via tyrosine phosphorylation as well, and that NLRP3 de-phosphorylation at specific tyrosine residues was required for inflammasome assembly and sustained IL-1β/IL-18 release. These findings demonstrated the importance of tyrosine phosphorylation as a key modulator of inflammasome activity. Following these initial reports, additional work elucidated that the activity of several inflammasome components is dictated via their phosphorylation status. Particularly, the action of specific tyrosine kinases and phosphatases are of critical importance for the regulation of inflammasome assembly and activity. By summarizing the currently available literature on the interaction of tyrosine phosphatases with inflammasome components we here provide an overview how tyrosine phosphatases affect the activation status of inflammasomes.

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Staurosporine inhibits agrin-induced acetylcholine receptor phosphorylation and aggregation.

The Journal of Cell Biology ◽

10.1083/jcb.125.3.661 ◽

1994 ◽

Vol 125 (3) ◽

pp. 661-668 ◽

Cited By ~ 64

Author(s):

B G Wallace

Keyword(s):

Tyrosine Phosphorylation ◽

Acetylcholine Receptor ◽

Tyrosine Kinases ◽

Neuromuscular Junctions ◽

Beta Subunit ◽

Dependent Manner ◽

Tyrosine Residues ◽

Serine Kinases ◽

Cultured Myotubes ◽

Dose Dependent

Agrin, a protein that mediates nerve-induced acetylcholine receptor (AChR) aggregation at developing neuromuscular junctions, has been shown to cause an increase in phosphorylation of the beta, gamma, and delta subunits of AChRs in cultured myotubes. As a step toward understanding the mechanism of agrin-induced AChR aggregation, we examined the effects of inhibitors of protein kinases on AChR aggregation and phosphorylation in chick myotubes in culture. Staurosporine, an antagonist of both protein serine and tyrosine kinases, blocked agrin-induced AChR aggregation in a dose-dependent manner; 50% inhibition occurred at approximately 2 nM. The extent of inhibition was independent of agrin concentration, suggesting an effect downstream of the interaction of agrin with its receptor. Staurosporine blocked agrin-induced phosphorylation of the AChR beta subunit, which occurs at least in part on tyrosine residues, but did not reduce phosphorylation of the gamma and delta subunits, which occurs on serine/threonine residues. Staurosporine also prevented the agrin-induced decrease in the rate at which AChRs are extracted from intact myotubes by mild detergents. H-7, an antagonist of protein serine kinases, inhibited agrin-induced phosphorylation of the gamma and delta subunits but did not block agrin-induced phosphorylation of the AChR beta subunit, AChR aggregation, or the decrease in AChR extractability. The results provide support for the hypothesis that tyrosine phosphorylation of the beta subunit plays a role in agrin-induced AChR aggregation.

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Endocytosis as a Mechanism for Tyrosine Kinase-dependent Suppression of a Voltage-gated Potassium Channel

Molecular Biology of the Cell ◽

10.1091/mbc.e03-11-0788 ◽

2004 ◽

Vol 15 (9) ◽

pp. 4073-4088 ◽

Cited By ~ 65

Author(s):

Edmund Nesti ◽

Brian Everill ◽

Anthony D. Morielli

Keyword(s):

Potassium Channel ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Physical Mechanism ◽

Ionic Current ◽

Dominant Negative ◽

Voltage Gated ◽

Dominant Negative Form ◽

N Terminus ◽

Negative Form

The voltage-gated potassium channel Kv1.2 undergoes tyrosine phosphorylation-dependent suppression of its ionic current. However, little is known about the physical mechanism behind that process. We have found that the Kv1.2 alpha-subunit protein undergoes endocytosis in response to the same stimuli that evoke suppression of Kv1.2 ionic current. The process is tyrosine phosphorylation-dependent because the same tyrosine to phenylalanine mutation in the N-terminus of Kv1.2 that confers resistance to channel suppression (Y132F) also confers resistance to channel endocytosis. Overexpression of a dominant negative form of dynamin blocked stimulus-induced Kv1.2 endocytosis and also blocked suppression of Kv1.2 ionic current. These data indicate that endocytosis of Kv1.2 from the cell surface is a key mechanism for channel suppression by tyrosine kinases.

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ZAP-70 binding specificity to T cell receptor tyrosine-based activation motifs: the tandem SH2 domains of ZAP-70 bind distinct tyrosine-based activation motifs with varying affinity.

Journal of Experimental Medicine ◽

10.1084/jem.181.1.375 ◽

1995 ◽

Vol 181 (1) ◽

pp. 375-380 ◽

Cited By ~ 133

Author(s):

N Isakov ◽

R L Wange ◽

W H Burgess ◽

J D Watts ◽

R Aebersold ◽

...

Keyword(s):

T Cell ◽

Tyrosine Phosphorylation ◽

T Cell Activation ◽

Tyrosine Kinases ◽

Cell Activation ◽

Sh2 Domain ◽

Critical Event ◽

Binding Studies ◽

Tyrosine Residues ◽

Sh2 Domains

Engagement of the T cell antigen receptor (TCR) results in activation of several tyrosine kinases leading to tyrosine phosphorylation of protein substrates and activation of multiple biochemical pathways. TCR-mediated activation of the src-family kinases, Lck and Fyn, results in tyrosine phosphorylation of the TCR zeta and CD3 chains. The site of phosphorylation in these chains is the tyrosine-based activation motif (TAM), a 15-16 amino acid module containing two tyrosine residues. Tyrosine-phosphorylated TAMs serve as targets for binding of the zeta-associated protein (ZAP-70) tyrosine kinase via its tandem SH2 domains. This binding correlates with activation of ZAP-70, a critical event in T cell activation. To further define the structural requirements for ZAP-70 interaction with the TCR, we developed a binding assay using immobilized glutathione S-transferase fusion proteins containing the NH2- and/or COOH-terminal SH2 domains of ZAP-70, and soluble synthetic peptides with the sequence of the cytoplasmic region of the TCR zeta chain (TCR zeta cyt) or individual TCR zeta and CD3 epsilon TAM motifs. Direct binding studies demonstrated that the tandem ZAP-70 SH2 domains bind phosphorylated, but not nonphosphorylated, TCR zeta cyt. The NH2-terminal ZAP-70 SH2 domain also binds to TCR zeta cyt but with 100-fold lower affinity. No binding was observed with the COOH-terminal ZAP-70 SH2 domain. Similar studies demonstrated that the ZAP-70 tandem SH2 domain can bind a TCR zeta 3 TAM peptide in which both tyrosine residues are phosphorylated: Little or no binding was observed with peptides phosphorylated at only one tyrosine residue, or a nonphosphorylated peptide. Binding of the tandem SH2 domains to the other two TCR zeta TAM peptides and to a CD3 epsilon TAM peptide was also observed. All four doubly tyrosine phosphorylated TAM peptides cross-compete with each other for binding to the tandem SH2 domains of ZAP-70. The affinity of these peptides for the tandem SH2 construct demonstrated a hierarchy of TAM zeta 1 > or = TAM zeta 2 > TAM epsilon > or = TAM zeta 3. The results provide further evidence that the ZAP-70 interaction with the TCR requires prior phosphorylation of both tyrosine residues within a TAM motif. Binding of ZAP-70 to phospho-TAMs is notable for the high level of cooperativity between the two SH2 domains, which individually demonstrate low affinity interaction with the ligand. The cooperativity ensures higher affinity for the doubly phosphorylated ligand. Affinity differences of as much as 30-fold indicates a significant specificity of interaction of ZAP-70 SH2 domains for different phospho-TAMs.

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Thrombopoietin stimulates cortactin translocation to the cytoskeleton independently of tyrosine phosphorylation

Biochemical Journal ◽

10.1042/bj3560875 ◽

2001 ◽

Vol 356 (3) ◽

pp. 875-881 ◽

Cited By ~ 8

Author(s):

Isabelle LOPEZ ◽

Véronique DUPREZ ◽

Josiane MELLE ◽

François DREYFUS ◽

Sylviane LÉVY-TOLÉDANO ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Kinase Inhibitor ◽

Shape Change ◽

Insoluble Fraction ◽

Actin Binding ◽

Syk Kinase ◽

Tyrosine Phosphorylated Protein ◽

Triton X ◽

Cortactin Tyrosine Phosphorylation ◽

Platelet Shape

Cortactin is an F-actin-binding protein expressed in platelets. During aggregation by thrombin, cortactin associates with Src, is tyrosine phosphorylated, and then translocates to the cytoskeleton. It is also found to associate with Syk during platelet shape change. Since cortactin undergoes tyrosine phosphorylation in platelets activated by thrombopoietin (TPO) that exhibit neither shape change nor aggregation, we investigated whether it could also relocalize to the detergent-insoluble fraction. We demonstrate that cortactin was present as a tyrosine-phosphorylated protein and co-localized with Syk in the Triton X-100-insoluble fraction of TPO-activated platelets. TPO stimulated Syk activation and association with cortactin. Conversely, cortactin associated with the kinases, Syk and Src. Cortactin tyrosine phosphorylation was blocked by Syk kinase inhibitor, piceatannol or Src family kinase inhibitor, PP2, suggesting that it depends on these two kinases. However, piceatannol or PP2 did not prevent cortactin translocation to the detergent-insoluble fraction. These data suggest that tyrosine phosphorylation is not required for cortactin translocation to the detergent-insoluble compartment. Furthermore, TPO activates, through its receptor c-Mpl, a signalling pathway to the cytoskeleton.

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Integrin-dependent translocation of LASP-1 to the cytoskeleton of activated platelets correlates with LASP-1 phosphorylation at tyrosine 171 by Src-kinase

Thrombosis and Haemostasis ◽

10.1160/th09-03-0143 ◽

2009 ◽

Vol 102 (09) ◽

pp. 520-528 ◽

Cited By ~ 16

Author(s):

Julia Traenka ◽

Christof R Hauck ◽

Urs Lewandrowski ◽

Albert Sickmann ◽

Stepan Gambaryan ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Phosphorylation Site ◽

Src Kinase ◽

Microscopic Analysis ◽

Leading Edge ◽

Site Directed Mutagenesis ◽

Actin Binding ◽

Intact Cells ◽

Integrin Αiibβ3

SummaryDuring platelet adhesion, the complex cytoskeletal structure is rearranged resulting in the formation of F-actin-based filopodia and lamellipodia. Stimulatory platelet signalling pathways include binding of integrin αIIbβ3 to fibrinogen followed by activation of protein tyrosine kinases (PTK) and phosphorylation of downstream signalling proteins. In this study, we demonstrate that the scaffolding and F-actin binding protein LASP-1 undergoes tyrosine phosphorylation in thrombin-stimulated human platelets. By means of specific inhibitors we identified Src-kinase as the primary enzyme phosphorylating LASP-1 in intact cells. These data were confirmed in platelet model cells (A5-CHO cells), constitutively expressing integrin αIIbβ3. Fibrinogen-mediated cell stimulation resulted in a similar tyrosine phosphorylation of transiently transfected LASP-1. Site directed mutagenesis identified tyrosine 171 as the Src-kinase phosphorylation site. Immunofluorescence microscopic analysis of these cells revealed a relocation of LASP-1 to focal contacts and the leading edge of the membrane upon fibrinogen activation and tyrosine 171 phosphorylation. This translocation was also seen in adherent platelets. Concomitant with adhesion, LASP-1 translocated from the cytosol along the arms of the pseudopodia into the leading lamellae of the spreading platelets, indicating a crucial role of the protein in platelet cytoskeleton rearrangement.

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Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III) Complexes

International Journal of Analytical Chemistry ◽

10.1155/2016/3216523 ◽

2016 ◽

Vol 2016 ◽

pp. 1-14 ◽

Cited By ~ 8

Author(s):

Jun Sumaoka ◽

Hiroki Akiba ◽

Makoto Komiyama

Keyword(s):

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

High Selectivity ◽

Protein Tyrosine Phosphatases ◽

Protein Tyrosine Kinases ◽

Selective Detection ◽

Protein Tyrosine ◽

Tyrosine Residues ◽

Recent Developments ◽

Efficient Screening

Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclearTbIIIcomplexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr), have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to theTbIIIion as the emission center. Even in the coexistence of phosphoserine (pSer) and phosphothreonine (pThr), pTyr can be efficintly detected with high selectivity. Simply by adding theseTbIIIcomplexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates.

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Upregulation of Cortactin Expression During the Maturation of Megakaryocytes

Blood ◽

10.1182/blood.v89.2.457 ◽

1997 ◽

Vol 89 (2) ◽

pp. 457-464 ◽

Cited By ~ 24

Author(s):

Xi Zhan ◽

Christian C. Haudenschild ◽

Yangson Ni ◽

Elizabeth Smith ◽

Cai Huang

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Bone Marrow Cells ◽

Actin Binding ◽

Specific Gene ◽

Related Protein ◽

Filamentous Actin ◽

Murine Bone Marrow

Abstract Cortactin is a potent filamentous actin-binding protein acting as a prominent substrate of Src tyrosine kinases. We have evaluated cortactin expression in a series of murine tissues and shown an abundant expression of cortactin in megakaryocytes and platelets. Cortactin, but not its related protein HS1, is upregulated during the phorbol 12-myristate 13-acetate (PMA)-mediated maturation of a human megakaryoblastic cell line CMK. Although the expression of Src-related kinases is also upregulated more rapidly than cortactin in PMA-treated CMK cells, tyrosine phosphorylation of cortactin appears to be only transiently elevated 4 days after PMA stimulation. In addition, cortactin expression is induced by thrombopoietin and interleukin-3 in megakaryocytes derived from murine bone marrow cells. Thus, cortactin represents a megakaryocyte-specific gene in bone marrow and the interaction of Src kinases with cortactin may be involved in the maturation of megakaryocytes.

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