RESUMO
A-kinase anchoring proteins (AKAPs) control the localization and substrate specificity of cAMP-dependent protein kinase (PKA), tetramers of regulatory (PKA-R) and catalytic (PKA-C) subunits, by binding to PKA-R subunits. Most mammalian AKAPs bind Type II PKA through PKA-RII (ref. 2), whereas dual specificity AKAPs bind both PKA-RI and PKA-RII (ref. 3). Inhibition of PKA-AKAP interactions modulates PKA signalling. Localized PKA activation in pseudopodia of migrating cells phosphorylates alpha4 integrins to provide spatial cues governing cell motility. Here, we report that the alpha4 cytoplasmic domain is a Type I PKA-specific AKAP that is distinct from canonical AKAPs in two ways: the alpha4 interaction requires the PKA holoenzyme, and is insensitive to amphipathic peptides that disrupt most PKA-AKAP interactions. We exploited type-specific PKA anchoring peptides to create genetically encoded baits that sequester specific PKA isoforms to the mitochondria and found that mislocalization of Type I, but not Type II, PKA disrupts alpha4 phosphorylation and markedly inhibits the velocity and directional persistence of cell migration.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Integrina alfa4/metabolismo , Animais , Western Blotting , Células CHO , Movimento Celular , Células Cultivadas , Cromatografia de Afinidade , Cricetinae , Cricetulus , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/genética , Cães , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação , Integrina alfa4/genética , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fosforilação , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In this study, we investigated the optical properties of a transition metal dichalcogenide (TMD) substrate via Mie-scattering-induced surface analysis (MISA). Employing near-field optical microscopy and finite-difference time-domain (FDTD) simulations, we systemically prove and directly visualize the Mie scattering of superspherical gold nanoparticles (s-AuNPs) at the nanoscale. Molybdenum disulfide substrates exhibited optical isotropy, while rhenium disulfide (ReS2) substrates showed anisotropic behavior attributed to the interaction with incident light's electric field. Our study revealed substantial anisotropic trends in Mie scattering, particularly in the near-infrared energy range, with ReS2 exhibiting more pronounced spectral and angular responses in satellite peaks. Our results emphasize the application of Mie scattering, exploring the optical properties of substrates and contributing to a deeper understanding of nanoscale light-matter interactions.
RESUMO
Considering that Korea's aging population is rapidly increasing, health serves as an indicator of older adults' quality of life, and dietary life directly affects their health. For health maintenance and improvement, preventive healthcare measures including safe food selection and nutritional supply are needed. This study aimed to evaluate the effect of senior-friendly diet on nutrition and health status improvement in older adults receiving community care. A total of 180 older adults were analyzed, with 154 and 26 in the senior-friendly diet intervention group and the general diet group, respectively. Surveys, blood tests, and frailty evaluations were conducted before and after the study. After 5 months of intervention, the blood status, nutrient intake, and frailty level were evaluated. The participants' mean age was 82.7 years, and 89.4% of them were living alone. In both groups, energy, protein, vitamin A, vitamin D, vitamin C, calcium, and magnesium intake were insufficient initially but generally improved after the intervention. Especially in the intervention group, energy, protein, vitamin D, vitamin C, and folic acid intake significantly increased. The frailty level also slightly improved, and the malnutrition rate was reduced. Even after the passage of time, the improvement effect size significantly differed between the groups. Therefore, resolving and supporting meals corresponding to the physiological needs of the older adults has a great impact on improving their quality of life, and such special consideration is a reasonable way to respond to a super-aged society.
Assuntos
Fragilidade , Estado Nutricional , Humanos , Idoso , Idoso de 80 Anos ou mais , Fragilidade/prevenção & controle , Fragilidade/epidemiologia , Vida Independente , Qualidade de Vida , Dieta , Vitamina D , Ácido Ascórbico , Idoso FragilizadoRESUMO
Formation of a stable lamellipodium at the front of migrating cells requires localization of Rac activation to the leading edge. Restriction of alpha4 integrin phosphorylation to the leading edge limits the interaction of alpha4 with paxillin to the sides and rear of a migrating cell. The alpha4-paxillin complex inhibits stable lamellipodia, thus confining lamellipod formation to the cell anterior. Here we report that binding of paxillin to the alpha4 integrin subunit inhibits adhesion-dependent lamellipodium formation by blocking Rac activation. The paxillin LD4 domain mediates this reduction in Rac activity by recruiting an ADP-ribosylation factor GTPase-activating protein (Arf-GAP) that decreases Arf activity, thereby inhibiting Rac. Finally, the localized formation of the alpha4-paxillin-Arf-GAP complex mediates the polarization of Rac activity and promotes directional cell migration. These findings establish a mechanism for the spatial localization of Rac activity to enhance cell migration.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Integrina alfa4/metabolismo , Integrina beta1/metabolismo , Fosfoproteínas/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Fatores de Ribosilação do ADP/efeitos dos fármacos , Animais , Células CHO , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/fisiologia , Cricetinae , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Ligantes , Substâncias Macromoleculares/metabolismo , Modelos Biológicos , Paxilina , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Pseudópodes/efeitos dos fármacos , Pseudópodes/fisiologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Cerebral cavernous malformation (CCM), a disease associated with defective endothelial junctions, result from autosomal dominant CCM1 mutations that cause loss of KRIT-1 protein function, though how the loss of KRIT-1 leads to CCM is obscure. KRIT-1 binds to Rap1, a guanosine triphosphatase that maintains the integrity of endothelial junctions. Here, we report that KRIT-1 protein is expressed in cultured arterial and venous endothelial cells and is present in cell-cell junctions. KRIT-1 colocalized and was physically associated with junctional proteins via its band 4.1/ezrin/radixin/moesin (FERM) domain. Rap1 activity regulated the junctional localization of KRIT-1 and its physical association with junction proteins. However, the association of the isolated KRIT-1 FERM domain was independent of Rap1. Small interfering RNA-mediated depletion of KRIT-1 blocked the ability of Rap1 to stabilize endothelial junctions associated with increased actin stress fibers. Thus, Rap1 increases KRIT-1 targeting to endothelial cell-cell junctions where it suppresses stress fibers and stabilizes junctional integrity.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Junções Intercelulares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Células CHO , Bovinos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Humanos , Proteína KRIT1 , Proteínas Associadas aos Microtúbulos/química , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas/químicaRESUMO
Dynamic regulation of adhesion complexes is required for cell migration and has therefore emerged as a key issue in the study of cell motility. Recent progress has been made in defining some of the molecular mechanisms by which adhesion disassembly is regulated, including the contributions of adhesion adaptor proteins and tyrosine kinases. However, little is known about the potential contribution of proteolytic mechanisms to the regulation of adhesion complex dynamics. Here, we show that proteolysis of talin by the intracellular calcium-dependent protease calpain is critical for focal adhesion disassembly. We have generated a single point mutation in talin that renders it resistant to proteolysis by calpain. Quantification of adhesion assembly and disassembly rates demonstrates that calpain-mediated talin proteolysis is a rate-limiting step during adhesion turnover. Furthermore, we demonstrate that disassembly of other adhesion components, including paxillin, vinculin and zyxin, is also dependent on the ability of calpain to cleave talin, suggesting a general role for talin proteolysis in regulating adhesion turnover. Together, these findings identify calpain-mediated proteolysis of talin as a mechanism by which adhesion dynamics are regulated.
Assuntos
Calpaína/metabolismo , Adesão Celular , Talina/metabolismo , Animais , Western Blotting , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Fibronectinas/metabolismo , Adesões Focais/metabolismo , Glicoproteínas/metabolismo , Humanos , Imuno-Histoquímica , Cinética , Camundongos , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Paxilina , Fosfoproteínas/metabolismo , Mutação Puntual , Testes de Precipitina , RNA Interferente Pequeno/metabolismo , Talina/genética , Vinculina/metabolismo , ZixinaRESUMO
The level of spatial accessibility is directly related to how street networks are connected. Connected or so-called "permeable" network systems encourage walking, cycling, and riding public transit. Fast urbanization during the recent decades in the world metropolises has created separated urban areas. Gated-style apartment complexes have led this segregation more obviously with their inaccessible internal networks. Opening the internal network of apartment complexes and redesigning the pedestrian paths among apartment buildings will significantly mitigate these networks' adverse effects on network permeability and increase spatial accessibility. This paper analyzes how such an opening design proposal for apartment complexes can change spatial accessibility using the case study of Mapo-gu, Seoul, Korea. It simulates three types of street networks and compares the results of accessibility in three conditions: (1) the internal networks of apartment complexes are not used by outsiders; (2) the internal networks of apartment complexes are open to outsiders with its existing entrances and path; and (3) the internal networks of sites are opened and redesigned by the Voronoi diagram method, which generates the optimal shortest path. An urban network analysis tool, Rhinoceros three-dimensional software, and Grashopper3D visual programming language have been used for the study results, which shows that a policy change in opening the intra-network of apartment complexes is likely to make the city more permeable. In addition, this study suggests extra modification on the pedestrian path for a higher level of accessibility in neighborhoods.
Assuntos
Planejamento Ambiental , Características de Residência , Cidades , República da Coreia , Seul , CaminhadaRESUMO
BACKGROUND: Integrin receptors, composed of transmembrane alpha and beta subunits, are essential for the development and functioning of multicellular animals. Agonist stimulation leads cells to regulate integrin affinity ("activation"), thus controlling cell adhesion and migration, controlling extracellular-matrix assembly, and contributing to angiogenesis, tumor cell metastasis, inflammation, the immune response, and hemostasis. A final step in integrin activation is the binding of talin, a cytoskeletal protein, to integrin beta cytoplasmic domains. Many different signaling molecules that regulate integrin affinity have been described, but a pathway that connects agonist stimulation to talin binding and activation has not been mapped. RESULTS: We used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in cells expressing a prototype activatable integrin, platelet alphaIIbbeta3. Phorbol myristate acetate (PMA) activated alphaIIbbeta3 only after the increased expression of both recombinant protein kinase Calpha (PKCalpha) and talin to levels approximating those in platelets. Inhibition of Rap1 GTPase reduced alphaIIbbeta3 activation, whereas activated Rap1A(G12V) bypassed the requirement for PKC, establishing that Rap1 is downstream of PKC. Talin binding to integrins mediates Rap1-induced activation because Rap1A(G12V) failed to activate alphaIIbbeta3 in cells expressing integrin binding-defective talin (W359A). Rap1 activated integrins by forming an integrin-associated complex containing talin in combination with the Rap effector, RIAM. Furthermore, siRNA-mediated knockdown of RIAM blocked integrin activation. CONCLUSIONS: We have, for the first time, ordered a pathway from agonist stimulation to integrin activation and established the Rap1-induced formation of an "integrin activation complex," containing RIAM and talin, that binds to and activates the integrin.
Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas/agonistas , Transdução de Sinais/fisiologia , Animais , Sítios de Ligação , Plaquetas/metabolismo , Células CHO , Cricetinae , Cricetulus , Proteínas de Fluorescência Verde/análise , Humanos , Modelos Biológicos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteína Quinase C/metabolismo , Proteínas Recombinantes de Fusão/análise , Transdução de Sinais/efeitos dos fármacos , Talina/metabolismo , Talina/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas rap1 de Ligação ao GTP/fisiologiaRESUMO
Antagonists to alpha4 integrin show promise for several autoimmune and inflammatory diseases but may exhibit mechanism-based toxicities. We tested the capacity of blockade of alpha4 integrin signaling to perturb functions involved in inflammation, while limiting potential adverse effects. We generated and characterized mice bearing a Y991A mutation in alpha4 integrin [alpha4(Y991A) mice], which blocks paxillin binding and inhibits alpha4 integrin signals that support leukocyte migration. In contrast to the embryonic-lethal phenotype of alpha4 integrin-null mice, mice bearing the alpha4(Y991A) mutation were viable and fertile; however, they exhibited defective recruitment of mononuclear leukocytes into thioglycollate-induced peritonitis. Alpha4 integrins are essential for definitive hematopoiesis; however, the alpha4(Y991A) mice had intact lymphohematopoiesis and, with the exception of reduced Peyer's patches, normal architecture and cellularity of secondary lymphoid tissues. We conclude that interference with alpha4 integrin signaling can selectively impair mononuclear leukocyte recruitment to sites of inflammation while sparing vital functions of alpha4 integrins in development and hematopoiesis.
Assuntos
Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Integrina alfa4/genética , Leucócitos Mononucleares/patologia , Paxilina/antagonistas & inibidores , Substituição de Aminoácidos/genética , Animais , Diferenciação Celular/fisiologia , Movimento Celular/genética , Feminino , Hematopoese/genética , Integrina alfa4/metabolismo , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Mutantes , Paxilina/metabolismo , Peritonite/metabolismo , Peritonite/patologia , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/crescimento & desenvolvimentoRESUMO
Integrins coordinate spatial signaling events essential for cell polarity and directed migration. Such signals from alpha4 integrins regulate cell migration in development and in leukocyte trafficking. Here, we report that efficient alpha4-mediated migration requires spatial control of alpha4 phosphorylation by protein kinase A, and hence localized inhibition of binding of the signaling adaptor, paxillin, to the integrin. In migrating cells, phosphorylated alpha4 accumulated along the leading edge. Blocking alpha4 phosphorylation by mutagenesis or by inhibition of protein kinase A drastically reduced alpha4-dependent migration and lamellipodial stability. alpha4 phosphorylation blocks paxillin binding in vitro; we now find that paxillin and phospho-alpha4 were in distinct clusters at the leading edge of migrating cells, whereas unphosphorylated alpha4 and paxillin colocalized along the lateral edges of those cells. Furthermore, enforced paxillin association with alpha4 inhibits migration and reduced lamellipodial stability. These results show that topographically specific integrin phosphorylation can control cell migration and polarization by spatial segregation of adaptor protein binding.
Assuntos
Movimento Celular/fisiologia , Integrina alfa4/metabolismo , Integrina alfa4beta1/metabolismo , Pseudópodes/metabolismo , Animais , Células CHO , Cricetinae , Proteínas do Citoesqueleto/metabolismo , Paxilina , Fosfoproteínas/metabolismo , Fosforilação , RatosRESUMO
BACKGROUND: The regeneration of peripheral nerve is associated with a change in the alternative splicing of the fibronectin primary gene transcript to re-express embryonic isoforms containing a binding site for alpha4beta1 integrins that promote neurite outgrowth. Here we use PC12 cells to examine the role of the interaction between paxillin and the alpha4 integrin cytoplasmic domain in neurite outgrowth. RESULTS: Expression of alpha4 with mutations in the paxillin-binding domain reduced neurite outgrowth on recombinant embryonic fibronectin fragments relative to wild type alpha4. Over-expression of paxillin promoted neurite outgrowth while a mutant isoform lacking the LD4 domain implicated in the regulation of ARF and Rac GTPases was less effective. Optimal alpha4-mediated migration in leucocytes requires spatial regulation of alpha4 phosphorylation at Ser988, a post-translational modification that blocks paxillin binding to the integrin cytoplasmic domain. In keeping with this alpha4(S988D), which mimics phosphorylated alpha4, did not promote neurite outgrowth. However, alpha4 was not phosphorylated in the PC12 cells, and a non-phosphorylatable alpha4(S988A) mutant promoted neurite outgrowth indistinguishably from the wild type integrin. CONCLUSION: We establish the importance of the alpha4 integrin-paxillin interaction in a model of axonal regeneration and highlight differing dependence on phosphorylation of alpha4 for extension of neuronal growth cones and migration of non-neural cells.
Assuntos
Integrina alfa4beta1/metabolismo , Regeneração Nervosa/fisiologia , Neuritos/fisiologia , Paxilina/metabolismo , Nervos Periféricos/crescimento & desenvolvimento , Animais , Movimento Celular/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Cones de Crescimento/metabolismo , Mutação , Regeneração Nervosa/genética , Fosforilação , RatosRESUMO
Activation of Rap1 small GTPases stabilizes cell--cell junctions, and this activity requires Krev Interaction Trapped gene 1 (KRIT1). Loss of KRIT1 disrupts cardiovascular development and causes autosomal dominant familial cerebral cavernous malformations. Here we report that native KRIT1 protein binds the effector loop of Rap1A but not H-Ras in a GTP-dependent manner, establishing that it is an authentic Rap1-specific effector. By modeling the KRIT1-Rap1 interface we designed a well-folded KRIT1 mutant that exhibited a ~40-fold-reduced affinity for Rap1A and maintained other KRIT1-binding functions. Direct binding of KRIT1 to Rap1 stabilized endothelial cell-cell junctions in vitro and was required for cardiovascular development in vivo. Mechanistically, Rap1 binding released KRIT1 from microtubules, enabling it to locate to cell--cell junctions, where it suppressed Rho kinase signaling and stabilized the junctions. These studies establish that the direct physical interaction of Rap1 with KRIT1 enables the translocation of microtubule-sequestered KRIT1 to junctions, thereby supporting junctional integrity and cardiovascular development.
Assuntos
Junções Intercelulares/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Musculares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Sistema Cardiovascular/crescimento & desenvolvimento , Sistema Cardiovascular/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Expressão Gênica , Vetores Genéticos , Células HEK293 , Humanos , Junções Intercelulares/metabolismo , Proteína KRIT1 , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno , Transdução de Sinais , Relação Estrutura-Atividade , Veias Umbilicais/citologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Proteínas rap1 de Ligação ao GTP/química , Proteínas rap1 de Ligação ao GTP/genéticaAssuntos
Proteínas de Ciclo Celular , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Proteínas Proto-Oncogênicas c-vav , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In mammals, beta1 integrin adhesion receptors generate signals that mediate cell spreading, migration, proliferation, and survival. CD98, a heterodimeric transmembrane protein, physically associates with certain integrin beta subunit cytoplasmic domains (tails) via its heavy chain, CD98hc (SLC3A2), and loss of CD98hc impairs integrin signaling. Here we have used the lack of CD98hc interaction with the Drosophila integrin betaPS tail for a homology scanning analysis that implicated the C-terminal 8 residues of beta3 (Thr(755)-Thr(802)) in CD98hc binding. We then identified point mutations in the beta3 C terminus (T755K and T758M) that abolish CD98hc association and a double mutation in the corresponding residues in the betaPS tail (K839T,M842T), which resulted in gain of CD98hc interaction. Furthermore, the loss of function beta3(T755K) mutation or the gain of function beta3/betaPS(K839T,M842T) led to a loss or gain of integrin-mediated cell spreading, respectively. Thus, we have identified critical integrin residues required for CD98hc interaction and in doing so have shown that CD98c interaction with the integrin beta tail is required for its ability to mediate integrin signaling. These studies also provide new insights into how CD98hc may cooperate with other cytoplasmic domain binding proteins to modulate integrin functions and into the evolution of integrin signaling.
Assuntos
Adesão Celular , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Cadeias beta de Integrinas/metabolismo , Animais , Células Cultivadas , Cadeia Pesada da Proteína-1 Reguladora de Fusão/fisiologia , Humanos , Cadeias beta de Integrinas/fisiologia , Integrina beta1/metabolismo , Integrina beta1/fisiologia , Integrina beta3/metabolismo , Integrina beta3/fisiologia , Camundongos , Mutagênese Sítio-Dirigida , Mutação Puntual , Transdução de SinaisRESUMO
Regulation of integrin affinity (activation) is essential for metazoan development and for many pathological processes. Binding of the talin phosphotyrosine-binding (PTB) domain to integrin beta subunit cytoplasmic domains (tails) causes activation, whereas numerous other PTB-domain-containing proteins bind integrins without activating them. Here we define the structure of a complex between talin and the membrane-proximal integrin beta3 cytoplasmic domain and identify specific contacts between talin and the integrin tail required for activation. We used structure-based mutagenesis to engineer talin and beta3 variants that interact with comparable affinity to the wild-type proteins but inhibit integrin activation by competing with endogenous talin. These results reveal the structural basis of talin's unique ability to activate integrins, identify an interaction that could aid in the design of therapeutics to block integrin activation, and enable engineering of cells with defects in the activation of multiple classes of integrins.
Assuntos
Integrinas/química , Integrinas/metabolismo , Talina/química , Talina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Proteínas de Ligação a DNA/metabolismo , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/químicaRESUMO
We used a TAP-tag approach to identify candidate binding proteins for the related Ras family GTPases: H-Ras, R-Ras, and Rap1A. Protein complexes were isolated from mouse fibroblasts, and component proteins were identified by a combination of nanoflow HPLC and tandem mass spectrometry. H-Ras was found to associate with numerous cytoskeletal proteins including talin-1. R-Ras and Rap1A each associated with various signaling molecules, many of which are membrane-associated. Thus, we have established the first database of potential Ras interactors in mammalian cells.
Assuntos
Cromatografia de Afinidade/métodos , Bases de Dados de Proteínas , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Camundongos , Células NIH 3T3 , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Proteínas ras/genéticaRESUMO
The alpha4 integrins (alpha4beta1 and alpha4beta7) play multiple roles in the immune system. Alpha4 integrins impact hematopoiesis, leukocyte trafficking in immune surveillance and inflammation, and leukocyte activation and survival. To perform these functions, alpha4 integrins act as both adhesive and signaling receptors. Paxillin, a signaling adapter molecule, binds directly to the alpha4 subunit cytoplasmic domain, and its binding is regulated by serine phosphorylation of the alpha4 subunit. This regulated interaction of paxillin with the alpha4 subunit is likely to regulate the diverse functions of alpha4 integrins in the immune system. Furthermore, this protein-protein interaction may provide novel targets for the modulation of the immune response.
Assuntos
Integrina alfa4/imunologia , Animais , Humanos , Integrina alfa4/metabolismo , Integrinas/imunologia , Integrinas/fisiologia , Ligação ProteicaRESUMO
alpha 4 integrins mediate increased cell migration and decreased cell spreading because the alpha 4 cytoplasmic domain (tail) binds tightly to paxillin, a signaling adaptor protein. Paxillin binding to the alpha 4 tail is blocked by alpha 4 phosphorylation at Ser988. To establish the biological role of alpha 4 phosphorylation, we reconstituted alpha 4-deficient Jurkat T cells with phosphorylation-mimicking (alpha 4(S988D)) or non-phosphorylatable (alpha 4(S988A)) mutants. alpha 4(S988D) disrupted paxillin binding and also inhibited cell migration and promoted cell spreading. In contrast, the non-phosphorylatable alpha 4(S988A) resulted in a further reduction in cell spreading; however, this mutation led to an unexpected suppression of cell migration. The suppression of cell migration by alpha 4(S988A) was ascribable to enhanced alpha 4-paxillin association, because enforced association by an alpha 4-paxillin fusion led to a phenotype similar to that of the non-phosphorylatable alpha 4(S988A) mutant. These data establish that optimal alpha 4-mediated cell migration requires both phosphorylation and dephosphorylation of the alpha 4 cytoplasmic domain to regulate the reversible binding of paxillin.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Integrina alfa4beta1/fisiologia , Fosfoproteínas/metabolismo , Linfócitos T/imunologia , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Moléculas de Adesão Celular/metabolismo , Membrana Celular/fisiologia , Cricetinae , Humanos , Células Jurkat , Mutagênese Sítio-Dirigida , Paxilina , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
We recently reported that CD47 (integrin-associated protein) on sickle red blood cells (SS RBCs) activates G-protein-dependent signaling, which promotes cell adhesion to immobilized thrombospondin (TSP) under relevant shear stress. These data suggested that signal transduction in SS RBCs may contribute to the vaso-occlusive pathology observed in sickle cell disease. However, the CD47-activated SS RBC adhesion receptor(s) that mediated adhesion to immobilized TSP remained unknown. Here we demonstrate that the alpha4beta1 integrin (VLA-4) is the receptor that mediates CD47-stimulated SS RBC adhesion to immobilized TSP. This adhesion requires both the N-terminal heparin-binding domain and the RGD site of TSP. CD47 signaling induces an "inside-out" activation of alpha4beta1 on SS RBCs as indicated by an RGD-dependent interaction of this integrin with soluble, plasma fibronectin. However, CD47 engagement also induces an alpha4beta1-mediated, RGD-independent adhesion of SS RBCs to immobilized vascular cell adhesion molecule-1 (VCAM-1). CD47 signaling in SS RBCs appears to be independent of large scale changes in cAMP formation but nonetheless promotes alpha4beta1-mediated adhesion via a protein kinase A-dependent, serine phosphorylation of the alpha4 cytoplasmic domain. CD47-activated SS RBC adhesion absolutely requires the Src family tyrosine kinases and is also enhanced by treatment of SS RBCs with low concentrations of cytochalasin D, which may release alpha4beta1 from cytoskeletal restraints. In addition, CD47 co-immunoprecipitates with alpha4beta1 in a sickle reticulocyte-enriched fraction of SS RBCs. These studies therefore identify the alpha4beta1 integrin on SS RBCs as a CD47-activated receptor for TSP, VCAM-1, and plasma fibronectin, revealing novel binding characteristics of this integrin.
Assuntos
Anemia Falciforme/sangue , Antígenos CD/metabolismo , Integrina alfa4beta1/metabolismo , Reticulócitos/metabolismo , Antígenos CD/química , Western Blotting , Antígeno CD47 , Adesão Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citocalasina D/farmacologia , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Fibronectinas/sangue , Fibronectinas/metabolismo , Humanos , Imunoprecipitação , Oligopeptídeos/química , Peptídeos/química , Fosforilação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Transdução de Sinais , Frações Subcelulares/metabolismo , Trombospondinas/metabolismo , Fatores de Tempo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Engagement of very late Ag-4 (integrin alpha(4)beta(1)) by ligands such as VCAM-1 markedly stimulates leukocyte migration mediated by LFA-1 (integrin alpha(L)beta(2)). This form of integrin trans-regulation in T cells requires the binding of paxillin to the alpha(4) integrin cytoplasmic domain. This conclusion is based on the abolition of trans-regulation in Jurkat T cells by an alpha(4) mutation (alpha(4)(Y991A)) that disrupts paxillin binding. Furthermore, cellular expression of an alpha(4)-binding fragment of paxillin that blocks the alpha(4)-paxillin interaction, selectively blocked VCAM-1 stimulation of alpha(L)beta(2)-dependent cell migration. The alpha(4)-paxillin association mediates trans-regulation by enhancing the activation of tyrosine kinases, focal adhesion kinase (FAK) and/or proline-rich tyrosine kinase-2 (Pyk2), based on two lines of evidence. First, disruption of the paxillin-binding site in the alpha(4) tail resulted in much less alpha(4)beta(1)-mediated phosphorylation of Pyk2 and FAK. Second, transfection with cDNAs encoding C-terminal fragments of Pyk2 and FAK, which block the function of the intact kinases, blocked alpha(4)beta(1) stimulation of alpha(L)beta(2)-dependent migration. These results define a proximal protein-protein interaction of an integrin cytoplasmic domain required for trans-regulation between integrins, and establish that augmented activation of Pyk2 and/or FAK is an immediate signaling event required for the trans-regulation of integrin alpha(L)beta(2) by alpha(4)beta(1).