scholarly journals Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin

2003 ◽  
Vol 160 (4) ◽  
pp. 565-575 ◽  
Author(s):  
Qiang Wang ◽  
Yi Xie ◽  
Quan-Sheng Du ◽  
Xiao-Jun Wu ◽  
Xu Feng ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Kinase ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Cell Periphery ◽  
Cytoskeletal Organization ◽  
Capping Protein ◽  
Tyrosine Kinase 2 ◽  
Multiple Cells ◽  
Actin Rings

Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Proline-rich tyrosine kinase 2 (PYK2), a major cell adhesion–activated tyrosine kinase in osteoclasts, plays an important role in regulating this event. The mechanisms by which PYK2 regulates actin cytoskeletal organization and osteoclastic activation remain largely unknown. In this paper, we provide evidence that PYK2 directly interacts with gelsolin, an actin binding, severing, and capping protein essential for osteoclastic actin cytoskeletal organization. The interaction is mediated via the focal adhesion–targeting domain of PYK2 and an LD motif in gelsolin's COOH terminus. PYK2 phosphorylates gelsolin at tyrosine residues and regulates gelsolin bioactivity, including decreasing gelsolin binding to actin monomer and increasing gelsolin binding to phosphatidylinositol lipids. In addition, PYK2 increases actin polymerization at the fibroblastic cell periphery. Finally, PYK2 interacts with gelsolin in osteoclasts, where PYK2 activation is required for the formation of actin rings. Together, our results suggest that PYK2 is a regulator of gelsolin, revealing a novel PYK2–gelsolin pathway in regulating actin cytoskeletal organization in multiple cells, including osteoclasts.

scholarly journals Regulation of Cdc42 Gtpase by Proline-Rich Tyrosine Kinase 2 Interacting with Psgap, a Novel Pleckstrin Homology and Src Homology 3 Domain Containing Rhogap Protein

2001 ◽  
Vol 152 (5) ◽  
pp. 971-984 ◽  
Author(s):  
Xiu-Rong Ren ◽  
Quan-Sheng Du ◽  
Yang-Zhong Huang ◽  
Shi-Zhou Ao ◽  
Lin Mei ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Pleckstrin Homology Domain ◽  
Cell Periphery ◽  
Dependent Manner ◽  
Pleckstrin Homology ◽  
Cytoskeletal Organization ◽  
Src Homology 3 ◽  
Tyrosine Kinase 2 ◽  
Src Homology 3 Domain ◽  
Src Homology

Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain–dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.

scholarly journals Differential Regulation of Proline-rich Tyrosine Kinase 2/Cell Adhesion Kinase β (PYK2/CAKβ) and pp125FAKby Glutamate and Depolarization in Rat Hippocampus

1996 ◽  
Vol 271 (46) ◽  
pp. 28942-28946 ◽  
Author(s):  
Julio C. Siciliano ◽  
Madeleine Toutant ◽  
Pascal Derkinderen ◽  
Terukatsu Sasaki ◽  
Jean-Antoine Girault
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Kinase ◽  
Differential Regulation ◽  
Rat Hippocampus ◽  
Tyrosine Kinase 2

scholarly journals Rickettsia Sca2 Recruits Two Actin Subunits for Nucleation but Lacks WH2 Domains

2018 ◽  
Author(s):  
SS Alqassim ◽  
IG Lee ◽  
R Dominguez
Keyword(s):  
Amino Acid ◽  
Actin Filament ◽  
Actin Polymerization ◽  
Intramolecular Interactions ◽  
Actin Binding ◽  
Globular Domain ◽  
Comet Tail ◽  
Capping Protein ◽  
High Affinity ◽  
Actin Nucleator

AbstractThe Rickettsia ~1,800 amino acid autotransporter protein Sca2 promotes actin polymerization on the surface of the bacterium to drive its movement using an actin comet tail mechanism. Sca2 mimics eukaryotic formins in that it promotes both actin filament nucleation and elongation and competes with capping protein to generate filaments that are long and unbranched. However, despite these functional similarities, Sca2 is structurally unrelated to eukaryotic formins and achieves these functions through an entirely different mechanism. Thus, while formins are dimeric, Sca2 functions as a monomer. However, Sca2 displays intramolecular interactions and functional cooperativity between its N- and C-terminal domains that are crucial for actin nucleation and elongation. Here, we map the interaction of N- and C-terminal fragments of Sca2 and their contributions to actin binding and nucleation. We find that both the N- and C-terminal regions of Sca2 interact with actin monomers, but only weakly, whereas the full-length protein binds two actin monomers with high affinity. Moreover, deletions at both ends of the N- and C-terminal regions disrupt their ability to interact with each other, suggesting that they form a contiguous ring-like structure that wraps around two actin subunits, analogous to the formin homology-2 (FH2) domain. The discovery of Sca2 as an actin nucleator followed the identification of what appeared to be a repeat of three WH2 domains in the middle of the molecule, consistent with the presence of WH2 domains in most actin nucleators. However, we show here that contrary to previous assumptions Sca2 does not contain WH2 domains, and that the corresponding region is folded as a globular domain that cooperates with other parts of the Sca2 molecule for actin binding and nucleation.

scholarly journals SRC-dependent outside-in signalling is a key step in the process of autoregulation of beta2 integrins in polymorphonuclear cells

2004 ◽  
Vol 380 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Paola PICCARDONI ◽  
Stefano MANARINI ◽  
Lorenzo FEDERICO ◽  
Zsuzsa BAGOLY ◽  
Romina PECCE ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Actin Polymerization ◽  
Protein Tyrosine Kinase ◽  
Polymorphonuclear Cells ◽  
High Avidity ◽  
Β2 Integrin ◽  
High Affinity ◽  
Tyrosine Kinase 2 ◽  
Oncogenic Protein ◽  
Β2 Integrins

In human PMN (polymorphonuclear cells), challenged by P-selectin, the β2-integrin Mac-1 (macrophage antigen-1) promoted the activation of the SRC (cellular homologue of Rous sarcoma virus oncogenic protein) family members HCK (haematopoietic cell kinase) and LYN (an SRC family protein tyrosine kinase) and phosphorylation of a P-110 (110 kDa protein). SRC kinase activity in turn was necessary for macrophage antigen-1-mediated adhesion [Piccardoni, Sideri, Manarini, Piccoli, Martelli, de Gaetano, Cerletti and Evangelista (2001) Blood 98, 108–116]. This suggested that an SRC-dependent outside-in signalling strengthens the β2-integrin interaction with the ligand. To support this hypothesis further, in the present study, we used the monoclonal antibody KIM127 or manganese to lock β2 integrins in a high-affinity state, and homotypic PMN adhesion was analysed to monitor β2-integrin adhesive function. KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation. Both these processes were abolished by blocking antibodies against the common β2 chain, by a combination of antibodies against αL and αM or by inhibitors of SRC activity. Confocal microscopy showed that activation epitopes were expressed by β2 integrins co-localized with patches of F-actin at the adhesion sites. Blockade of SRC kinases or of actin polymerization prevented clustering of activated integrins as well as F-actin accumulation. FACS analysis showed that SRC inhibitors modified neither basal nor manganese-induced KIM127 binding. An SRC-dependent outside-in signalling initiated by β2 integrins was also required for adhesion triggered by interleukin-8. These results confirm the hypothesis that an SRC-dependent outside-in signalling triggered by high affinity and ligand binding is necessary to stabilize β2-integrin-mediated adhesion. Allowing clustering of activated integrins, SRC might link the high-affinity with the high-avidity state. Proline-rich tyrosine kinase-2 appears to be involved in this process.

scholarly journals Tyrosyl Phosphorylated PAK1 Regulates Breast Cancer Cell Motility in Response to Prolactin through Filamin A

2013 ◽  
Vol 27 (3) ◽  
pp. 455-465 ◽  
Author(s):  
Alan Hammer ◽  
Leah Rider ◽  
Peter Oladimeji ◽  
Leslie Cook ◽  
Quanwen Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tyrosine Kinase ◽  
Cell Motility ◽  
Small Gtpase ◽  
Actin Binding ◽  
Filamin A ◽  
T47d Cells ◽  
Tyrosine Kinase 2 ◽  
Janus Tyrosine Kinase 2

Abstract The p21-activated serine-threonine kinase (PAK1) is activated by small GTPase-dependent and -independent mechanisms and regulates cell motility. Both PAK1 and the hormone prolactin (PRL) have been implicated in breast cancer by numerous studies. We have previously shown that the PRL-activated tyrosine kinase JAK2 (Janus tyrosine kinase 2) phosphorylates PAK1 in vivo and identified tyrosines (Tyr) 153, 201, and 285 in the PAK1 molecule as sites of JAK2 tyrosyl phosphorylation. Here, we have used human breast cancer T47D cells stably overexpressing PAK1 wild type or PAK1 Y3F mutant in which Tyr(s) 153, 201, and 285 were mutated to phenylalanines to demonstrate that phosphorylation of these three tyrosines are required for maximal PRL-dependent ruffling. In addition, phosphorylation of these three tyrosines is required for increased migration of T47D cells in response to PRL as assessed by two independent motility assays. Finally, we show that PAK1 phosphorylates serine (Ser) 2152 of the actin-binding protein filamin A to a greater extent when PAK1 is tyrosyl phosphorylated by JAK2. Down-regulation of PAK1 or filamin A abolishes the effect of PRL on cell migration. Thus, our data presented here bring some insight into the mechanism of PRL-stimulated motility of breast cancer cells.

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.

A Pyk2–Vav1 complex is recruited to β3-adhesion sites to initiate Rho activation

2009 ◽  
Vol 420 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Chunlei Gao ◽  
Scott D. Blystone
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Fluorescent Protein ◽  
Rho Gtpase ◽  
Adaptor Protein ◽  
Direct Interaction ◽  
Nucleotide Exchange ◽  
Tyrosine Kinase 2

Integrin αvβ3-mediated adhesion of haemopoietic cells to vitronectin results in β3 tyrosine phosphorylation and Rho activation which is necessary for adhesion. Previously, we have shown that the RhoGEF (Rho guanine-nucleotide-exchange factor) Vav1 could associate indirectly with αvβ3 during leucocyte adhesion to vitronectin. In the present study, we have identified the non-receptor tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) as the adaptor protein that links Vav1 with αvβ3. The association of Pyk2 and Vav1 with β3 relies on the presence of Tyr747 in β3, the primary site of β3 phosphorylation. However, association of Pyk2 with Vav1 is independent of β3 tyrosine phosphorylation. Formation of a Pyk2–Vav1 complex occurs upon cell adhesion and Pro717 of Pyk2 plays a key role in Pyk2 interaction with Vav1. Utilizing purified recombinant proteins, we confirmed the direct interaction between Pyk2 and Vav1 In vitro. Cells transfected with GFP (green fluorescent protein)–Pyk2-P717A demonstrated severely suppressed cytoskeletal reorganization, impaired Vav1 recruitment, decreased Rho GTPase activation and loss of cell adhesion. Using siRNA (small interfering RNA) to specifically reduce Pyk2 levels in cells resulted in disrupted association between Vav1 and β3 and impaired cell adhesion. These results indicate that Pyk2 is a critical signalling molecule downstream of β3 integrin tyrosine phosphorylation and mediates Vav1 recruitment to accomplish actin reorganization necessary for adhesion.

scholarly journals Thymosin β4 is essential for adherens junction stability and epidermal planar cell polarity

Development ◽  
2020 ◽  
Vol 147 (23) ◽  
pp. dev193425
Author(s):  
Krishnanand Padmanabhan ◽  
Hanna Grobe ◽  
Jonathan Cohen ◽  
Arad Soffer ◽  
Adnan Mahly ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Polarity ◽  
Actin Polymerization ◽  
Planar Cell Polarity ◽  
Adherens Junction ◽  
Actin Binding ◽  
Thymosin Β4 ◽  
Eyelid Closure

ABSTRACTPlanar cell polarity (PCP) is essential for tissue morphogenesis and homeostasis; however, the mechanisms that orchestrate the cell shape and packing dynamics required to establish PCP are poorly understood. Here, we identified a major role for the globular (G)-actin-binding protein thymosin-β4 (TMSB4X) in PCP establishment and cell adhesion in the developing epidermis. Depletion of Tmsb4x in mouse embryos hindered eyelid closure and hair-follicle angling owing to PCP defects. Tmsb4x depletion did not preclude epidermal cell adhesion in vivo or in vitro; however, it resulted in abnormal structural organization and stability of adherens junction (AJ) due to defects in filamentous (F)-actin and G-actin distribution. In cultured keratinocytes, TMSB4X depletion increased the perijunctional G/F-actin ratio and decreased G-actin incorporation into junctional actin networks, but it did not change the overall actin expression level or cellular F-actin content. A pharmacological treatment that increased the G/F-actin ratio and decreased actin polymerization mimicked the effects of Tmsb4x depletion on both AJs and PCP. Our results provide insights into the regulation of the actin pool and its involvement in AJ function and PCP establishment.

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