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

2003 ◽  
Vol 14 (8) ◽  
pp. 3216-3229 ◽  
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.

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

2004 ◽  
Vol 380 (2) ◽  
pp. 581-591 ◽  
Author(s):  
Lingzhi FAN ◽  
Caterina Di CIANO-OLIVEIRA ◽  
Scott A. WEED ◽  
Andrew W. B. CRAIG ◽  
Peter A. GREER ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Feedback Mechanism ◽  
Reciprocal Relationship ◽  
Actin Binding ◽  
Hypertonic Stress ◽  
Tyrosine Residues ◽  
Actin Depolymerization ◽  
N Terminus ◽  
Cortactin Tyrosine Phosphorylation

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.

Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-gamma in quiescent BALB/c 3T3 cells

1989 ◽  
Vol 9 (7) ◽  
pp. 2934-2943
Author(s):  
M I Wahl ◽  
N E Olashaw ◽  
S Nishibe ◽  
S G Rhee ◽  
W J Pledger ◽  
...  
Keyword(s):  
Growth Factor ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Hormonal Regulation ◽  
Growth Factor Receptor ◽  
Fold Increase ◽  
Platelet Derived Growth Factor ◽  
3T3 Cells ◽  
Pdgf Stimulation

Platelet-derived growth factor (PDGF) stimulates the proliferation of quiescent fibroblasts through a series of events initiated by activation of tyrosine kinase activity of the PDGF receptor at the cell surface. Physiologically significant substrates for this or other growth factor receptor or oncogene tyrosine kinases have been difficult to identify. Phospholipase C (PLC), a key enzyme of the phosphoinositide pathway, is believed to be an important site for hormonal regulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces the intracellular second-messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Treatment of BALB/c 3T3 cells with PDGF led to a rapid (within 1 min) and significant (greater than 50-fold) increase in PLC activity, as detected in eluates of proteins from a phosphotyrosine immunoaffinity matrix. This PDGF-stimulated increase in phosphotyrosine-immunopurified PLC activity occurred for up to 12 h after addition of growth factor to quiescent cells. Interestingly, the PDGF stimulation occurred at 3 as well as 37 degrees C and in the absence or presence of extracellular Ca2+. Immunoprecipitation of cellular proteins with monoclonal antibodies specific for three distinct cytosolic PLC isozymes demonstrated the presence of a 145-kilodalton isozyme, PLC-gamma (formerly PLC-II), in BALB/c 3T3 cells. Furthermore, these immunoprecipitation studies showed that PLC-gamma is rapidly phosphorylated on tyrosine residues after PDGF stimulation. The results suggest that mitogenic signaling by PDGF is coincident with tyrosine phosphorylation of PLC-gamma.

Internalization of activated platelet-derived growth factor receptor-phosphatidylinositol-3' kinase complexes: potential interactions with the microtubule cytoskeleton

1993 ◽  
Vol 13 (10) ◽  
pp. 6052-6063
Author(s):  
R Kapeller ◽  
R Chakrabarti ◽  
L Cantley ◽  
F Fay ◽  
S Corvera
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Endocytic Pathway ◽  
Platelet Derived Growth Factor ◽  
Cell Periphery ◽  
Perinuclear Region ◽  
High Concentration ◽  
Microtubule Cytoskeleton ◽  
Microtubule Network

Phosphatidylinositol (PI)-3' kinase catalyzes the formation of PI 3,4-diphosphate and PI 3,4,5-triphosphate in response to stimulation of cells by platelet-derived growth factor (PDGF). Here we report that tyrosine-phosphorylated PDGF receptors, the p85 subunit of PI-3' kinase (p85), and activated PI-3' kinase are found in isolated clathrin-coated vesicles within 2 min of exposure of cells to PDGF, indicating that both receptor and activated PI-3' kinase enter the endocytic pathway. Immunofluorescence analysis of p85 in serum-starved cells revealed a punctate/reticular staining pattern, concentrated in the perinuclear region and displaying high focal concentration at the centrosome. In addition, partial coalignment of p85 with microtubules was observed after optical sectioning microscopy and image reconstruction. The association of p85 with the microtubule network was further evidenced by the microtubule-depolymerizing drug nocodazole, which caused a redistribution of p85 from the perinuclear region to the cell periphery. Interestingly, the most significant effect of PDGF on the distribution of p85 was an increase in the staining intensity of this protein in the perinuclear region, and this effect was eliminated by prior treatment of cells with nocodazole. These results suggest that PDGF receptor-p85 complexes internalize and transit in association with the microtubule cytoskeleton. In addition, the high concentration of p85 in intracellular structures in the absence of PDGF stimulation suggests additional roles for this protein independent of its association with receptor tyrosine kinases.

Filling the GAPs in cell dynamics control: BPGAP1 promotes cortactin translocation to the cell periphery for enhanced cell migration

2004 ◽  
Vol 32 (6) ◽  
pp. 1110-1112 ◽  
Author(s):  
B.L. Lua ◽  
B.C. Low
Keyword(s):  
Cell Migration ◽  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Small Gtpases ◽  
Cell Periphery ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Cell Dynamics ◽  
Cellular Division

Cells undergo dynamic changes in morphology or motility during cellular division and proliferation, differentiation, neuronal pathfinding, wound healing, apoptosis, host defense and organ development. These processes are controlled by signalling events relayed through cascades of protein interactions leading to the establishment and maintenance of cytoskeletal networks of microtubules and actin. Various regulators, including the Rho small GTPases (guanine nucleotide triphosphatases), serve as master switches to fine-tune the amplitude, duration as well as the integration of such circuitry responses. Rho GTPases are activated by guanine nucleotide-exchange factors and inactivated by GAPs (GTPase-activating proteins). Although normally down-regulating signalling pathways by catalysing their GTPase activity, many GAPs exist with various protein modules, the functions of which still largely remain unknown. BPGAP1 is a novel RhoGAP that co-ordinately regulates pseudopodia and cell migration through the interplay of its BNIP-2 and Cdc42GAP homology domains serving as a homophilic/heterophilic interaction device, an enzymic RhoGAP domain that inactivates RhoA and a proline-rich region that binds the Src homology-3 domain of cortactin. Both proteins co-localize to cell periphery and enhance cell migration. As a molecular scaffold in cortical actin assembly and organization, cortactin and its interaction with small GTPases, GAPs and tyrosine kinases seems set to provide further insights to the multiplicity and complexity of cell dynamics control. Elucidating how these processes might be individually or co-ordinately regulated through cortactin remains an exciting future challenge.

scholarly journals Actin-depolymerizing Factor and Cofilin-1 Play Overlapping Roles in Promoting Rapid F-Actin Depolymerization in Mammalian Nonmuscle Cells

2005 ◽  
Vol 16 (2) ◽  
pp. 649-664 ◽  
Author(s):  
Pirta Hotulainen ◽  
Eija Paunola ◽  
Maria K. Vartiainen ◽  
Pekka Lappalainen
Keyword(s):  
Cell Motility ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Depolymerizing Factor ◽  
Cytoskeletal Dynamics ◽  
Nonmuscle Cells ◽  
In Cells

Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling “old” actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.

scholarly journals Critical Roles of Phosphorylation and Actin Binding Motifs, but Not the Central Proline-rich Region, for Ena/Vasodilator-stimulated Phosphoprotein (VASP) Function during Cell Migration

2002 ◽  
Vol 13 (7) ◽  
pp. 2533-2546 ◽  
Author(s):  
Joseph J. Loureiro ◽  
Douglas A. Rubinson ◽  
James E. Bear ◽  
Gretchen A. Baltus ◽  
Adam V. Kwiatkowski ◽  
...  
Keyword(s):  
Cell Motility ◽  
Cell Movement ◽  
Protein Family ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Binding Motif ◽  
Conserved Sequence ◽  
Cellular Processes ◽  
Sequence Elements

The Ena/vasodilator-stimulated phosphoprotein (VASP) protein family is implicated in the regulation of a number of actin-based cellular processes, including lamellipodial protrusion necessary for whole cell translocation. A growing body of evidence derived largely from in vitro biochemical experiments using purified proteins, cell-free extracts, and pathogen motility has begun to suggest various mechanistic roles for Ena/VASP proteins in the control of actin dynamics. Using complementation of phenotypes in Ena/VASP-deficient cells and overexpression in normal fibroblasts, we have assayed the function of a panel of mutants in one member of this family, Mena, by mutating highly conserved sequence elements found in this protein family. Surprisingly, deletion of sites required for binding of the actin monomer-binding protein profilin, a known ligand of Ena/VASP proteins, has no effect on the ability of Mena to regulate random cell motility. Our analysis revealed two features essential for Ena/VASP function in cell movement, cyclic nucleotide-dependent kinase phosphorylation sites and an F-actin binding motif. Interestingly, expression of the C-terminal EVH2 domain alone is sufficient to complement loss of Ena/VASP function in random cell motility.

scholarly journals Characterization of Integrin–Tetraspanin Adhesion Complexes

1999 ◽  
Vol 146 (2) ◽  
pp. 477-492 ◽  
Author(s):  
Fedor Berditchevski ◽  
Elena Odintsova
Keyword(s):  
Actin Cytoskeleton ◽  
Tyrosine Phosphorylation ◽  
Cellular Localization ◽  
Protein Complexes ◽  
Ectopic Expression ◽  
Cell Periphery ◽  
Integrin Receptors ◽  
Cortical Actin ◽  
In Cells

Tetraspanins (or proteins from the transmembrane 4 superfamily, TM4SF) form membrane complexes with integrin receptors and are implicated in integrin-mediated cell migration. Here we characterized cellular localization, structural composition, and signaling properties of α3β1–TM4SF adhesion complexes. Double-immunofluorescence staining showed that various TM4SF proteins, including CD9, CD63, CD81, CD82, and CD151 are colocalized within dot-like structures that are particularly abundant at the cell periphery. Differential extraction in conjunction with chemical cross-linking indicated that the cell surface fraction of α3β1–TM4SF protein complexes may not be directly linked to the cytoskeleton. However, in cells treated with cytochalasin B α3β1–TM4SF protein complexes are relocated into intracellular vesicles suggesting that actin cytoskeleton plays an important role in the distribution of tetraspanins into adhesion structures. Talin and MARCKS are partially codistributed with TM4SF proteins, whereas vinculin is not detected within the tetraspanin-containing adhesion structures. Attachment of serum-starved cells to the immobilized anti-TM4SF mAbs induced dephosphorylation of focal adhesion kinase (FAK). On the other hand, clustering of tetraspanins in cells attached to collagen enhanced tyrosine phosphorylation of FAK. Furthermore, ectopic expression of CD9 in fibrosarcoma cells affected adhesion-induced tyrosine phosphorylation of FAK, that correlated with the reorganization of the cortical actin cytoskeleton. These results show that tetraspanins can modulate integrin signaling, and point to a mechanism by which TM4SF proteins regulate cell motility.

scholarly journals Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling

Blood ◽  
2007 ◽  
Vol 110 (12) ◽  
pp. 4111-4119 ◽  
Author(s):  
Linlin Wang ◽  
Thomas C. Schulz ◽  
Eric S. Sherrer ◽  
Derek S. Dauphin ◽  
Soojung Shin ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Embryonic Stem ◽  
Growth Factor Receptor ◽  
Insulin Like Growth Factor ◽  
Fibroblast Growth ◽  
Fibroblast Growth Factor Receptors ◽  
Self Renewal

Abstract Despite progress in developing defined conditions for human embryonic stem cell (hESC) cultures, little is known about the cell-surface receptors that are activated under conditions supportive of hESC self-renewal. A simultaneous interrogation of 42 receptor tyrosine kinases (RTKs) in hESCs following stimulation with mouse embryonic fibroblast (MEF) conditioned medium (CM) revealed rapid and prominent tyrosine phosphorylation of insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R); less prominent tyrosine phosphorylation of epidermal growth factor receptor (EGFR) family members, including ERBB2 and ERBB3; and trace phosphorylation of fibroblast growth factor receptors. Intense IGF1R and IR phosphorylation occurred in the absence of MEF conditioning (NCM) and was attributable to high concentrations of insulin in the proprietary KnockOut Serum Replacer (KSR). Inhibition of IGF1R using a blocking antibody or lentivirus-delivered shRNA reduced hESC self-renewal and promoted differentiation, while disruption of ERBB2 signaling with the selective inhibitor AG825 severely inhibited hESC proliferation and promoted apoptosis. A simple defined medium containing an IGF1 analog, heregulin-1β (a ligand for ERBB2/ERBB3), fibroblast growth factor-2 (FGF2), and activin A supported long-term growth of multiple hESC lines. These studies identify previously unappreciated RTKs that support hESC proliferation and self-renewal, and provide a rationally designed medium for the growth and maintenance of pluripotent hESCs.

scholarly journals Essential role of MARCKS in cortical actin dynamics during gastrulation movements

2004 ◽  
Vol 164 (2) ◽  
pp. 169-174 ◽  
Author(s):  
Hidekazu Iioka ◽  
Naoto Ueno ◽  
Noriyuki Kinoshita
Keyword(s):  
Wnt Pathway ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Convergent Extension ◽  
Embryonic Cells ◽  
Cortical Actin ◽  
Morphogenetic Movements ◽  
Kinase Substrate ◽  
Cytoskeletal Regulation

Myristoylated alanine-rich C kinase substrate (MARCKS) is an actin-binding, membrane-associated protein expressed during Xenopus embryogenesis. We analyzed its function in cytoskeletal regulation during gastrulation. Here, we show that blockade of its function impaired morphogenetic movements, including convergent extension. MARCKS was required for control of cell morphology, motility, adhesion, protrusive activity, and cortical actin formation in embryonic cells. We also demonstrate that the noncanonical Wnt pathway promotes the formation of lamellipodia- and filopodia-like protrusions and that MARCKS is necessary for this activity. These findings show that MARCKS regulates the cortical actin formation that is requisite for dynamic morphogenetic movements.

scholarly journals Cortactin Localization to Sites of Actin Assembly in Lamellipodia Requires Interactions with F-Actin and the Arp2/3 Complex

2000 ◽  
Vol 151 (1) ◽  
pp. 29-40 ◽  
Author(s):  
Scott A. Weed ◽  
Andrei V. Karginov ◽  
Dorothy A. Schafer ◽  
Alissa M. Weaver ◽  
Andrew W. Kinley ◽  
...  
Keyword(s):  
Tandem Repeat ◽  
Actin Polymerization ◽  
Spatial Organization ◽  
Pdz Domain ◽  
Actin Binding ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Amino Terminal ◽  
Receptor Complexes

Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Here, we report that cortactin is localized with the actin-related protein (Arp) 2/3 complex at sites of actin polymerization within the lamellipodia. Two distinct sequence motifs of cortactin contribute to its interaction with the cortical actin network: the fourth of six tandem repeats and the amino-terminal acidic region (NTA). Cortactin variants lacking either the fourth tandem repeat or the NTA failed to localize at the cell periphery. Tandem repeat four was necessary for cortactin to stably bind F-actin in vitro. The NTA region interacts directly with the Arp2/3 complex based on affinity chromatography, immunoprecipitation assays, and binding assays using purified components. Cortactin variants containing the NTA region were inefficient at promoting Arp2/3 actin nucleation activity. These data provide strong evidence that cortactin is specifically localized to sites of dynamic cortical actin assembly via simultaneous interaction with F-actin and the Arp2/3 complex. Cortactin interacts via its Src homology 3 (SH3) domain with ZO-1 and the SHANK family of postsynaptic density 95/dlg/ZO-1 homology (PDZ) domain–containing proteins, suggesting that cortactin contributes to the spatial organization of sites of actin polymerization coupled to selected cell surface transmembrane receptor complexes.

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