The activation of Src kinase in cells is strictly controlled by intramolecular inhibitory interactions mediated by SH3 and SH2 domains. They impose structural constraints on the kinase domain holding it in a catalytically non-permissive state. The transition between inactive and active conformation is known to be largely regulated by the phosphorylation state of key tyrosines 416 and 527. Here, we identified that phosphorylation of tyrosine 90 reduces binding affinity of the SH3 domain to its interacting partners, opens the Src structure, and renders Src catalytically active. This is accompanied by an increased affinity to the plasma membrane, decreased membrane motility, and slower diffusion from focal adhesions. Phosphorylation of tyrosine 90 controlling SH3-medited intramolecular inhibitory interaction, analogical to tyrosine 527 regulating SH2-C-terminus bond, enables SH3 and SH2 domains to serve as cooperative but independent regulatory elements. This mechanism allows Src to adopt several distinct conformations of varying catalytic activities and interacting properties, enabling it to operate not as a simple switch but as a tunable regulator functioning as a signalling hub in a variety of cellular processes.

• Klíčová slova
• SH3 domain, Src, biochemistry, cell biology, cell transformation, chemical biology, invasiveness, mouse, phosphorylation, protein structure,
• MeSH
• fosforylace MeSH
• skupina kinas odvozených od src-genu * metabolismus   MeSH
• src homologní domény * MeSH
• tyrosin metabolismus   MeSH
• tyrosinkinasy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• skupina kinas odvozených od src-genu * MeSH
• tyrosin MeSH
• tyrosinkinasy MeSH

MT1-MMP (MMP-14) is a multifunctional protease that regulates ECM degradation, activation of other proteases, and a variety of cellular processes, including migration and viability in physiological and pathological contexts. Both the localization and signal transduction capabilities of MT1-MMP are dependent on its cytoplasmic domain that constitutes the final 20 C-terminal amino acids, while the rest of the protease is extracellular. In this review, we summarize the ways in which the cytoplasmic tail is involved in regulating and enacting the functions of MT1-MMP. We also provide an overview of known interactors of the MT1-MMP cytoplasmic tail and the functional significance of these interactions, as well as further insight into the mechanisms of cellular adhesion and invasion that are regulated by the cytoplasmic tail.

• Klíčová slova
• MT1-MMP, cell invasion, intracellular trafficking, matrix metalloproteinases, post-translational modifications,
• MeSH
• buněčná adheze MeSH
• matrixová metaloproteinasa 14 * metabolismus   MeSH
• pohyb buněk MeSH
• signální transdukce * MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• matrixová metaloproteinasa 14 * MeSH

Protein p130Cas constitutes an adaptor protein mainly involved in integrin signaling downstream of Src kinase. Owing to its modular structure, p130Cas acts as a general regulator of cancer cell growth and invasiveness induced by different oncogenes. However, other mechanisms of p130Cas signaling leading to malignant progression are poorly understood. Here, we show a novel interaction of p130Cas with Ser/Thr kinase PKN3, which is implicated in prostate and breast cancer growth downstream of phosphoinositide 3-kinase. This direct interaction is mediated by the p130Cas SH3 domain and the centrally located PKN3 polyproline sequence. PKN3 is the first identified Ser/Thr kinase to bind and phosphorylate p130Cas and to colocalize with p130Cas in cell structures that have a pro-invasive function. Moreover, the PKN3-p130Cas interaction is important for mouse embryonic fibroblast growth and invasiveness independent of Src transformation, indicating a mechanism distinct from that previously characterized for p130Cas. Together, our results suggest that the PKN3-p130Cas complex represents an attractive therapeutic target in late-stage malignancies.

• Klíčová slova
• CAS, BCAR1, PKN3, SH3, Src, p130Cas,
• MeSH
• fibroblasty metabolismus   MeSH
• fosforylace MeSH
• fosfothreonin metabolismus   MeSH
• invazivní růst nádoru MeSH
• kontraktilní svazky metabolismus   MeSH
• lidé MeSH
• myši nahé MeSH
• nádory metabolismus   patologie   MeSH
• podozomy metabolismus   MeSH
• pohyb buněk MeSH
• proliferace buněk MeSH
• proteinkinasa C metabolismus   MeSH
• pseudopodia metabolismus   MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• substrátový protein asociovaný s Crk metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfothreonin MeSH
• protein kinase N MeSH Prohlížeč
• proteinkinasa C MeSH
• skupina kinas odvozených od src-genu MeSH
• substrátový protein asociovaný s Crk MeSH

CAS is a docking protein downstream of the proto-oncogene Src with a role in invasion and metastasis of cancer cells. The CAS SH3 domain is indispensable for CAS-mediated signaling, but structural aspects of CAS SH3 ligand binding and regulation are not well understood. Here, we identified the consensus CAS SH3 binding motif and structurally characterized the CAS SH3 domain in complex with ligand. We revealed the requirement for an uncommon centrally localized lysine residue at position +2 of CAS SH3 ligands and two rather dissimilar optional anchoring residues, leucine and arginine, at position +5. We further expanded the knowledge of CAS SH3 ligand binding regulation by manipulating tyrosine 12 phosphorylation and confirmed the negative role of this phosphorylation on CAS SH3 ligand binding. Finally, by exploiting the newly identified binding requirements of the CAS SH3 domain, we predicted and experimentally verified two novel CAS SH3 binding partners, DOK7 and GLIS2.

• MeSH
• aminokyseliny metabolismus   MeSH
• fosforylace fyziologie   MeSH
• lidé MeSH
• ligandy MeSH
• protoonkogen Mas MeSH
• sekvence aminokyselin MeSH
• signální transdukce fyziologie   MeSH
• src homologní domény fyziologie   MeSH
• substrátový protein asociovaný s Crk metabolismus   MeSH
• vazba proteinů fyziologie   MeSH
• vazebná místa fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• ligandy MeSH
• MAS1 protein, human MeSH Prohlížeč
• protoonkogen Mas MeSH
• substrátový protein asociovaný s Crk MeSH

CAS is a docking protein, which was shown to act as a mechanosensor in focal adhesions. The unique assembly of structural domains in CAS is important for its function as a mechanosensor. The tension within focal adhesions is transmitted to a stretchable substrate domain of CAS by focal adhesion-targeting of SH3 and CCH domain of CAS, which anchor the CAS protein in focal adhesions. Mechanistic models of the stretching biosensor propose equal roles for both anchoring domains. Using deletion mutants and domain replacements, we have analyzed the relative importance of the focal adhesion anchoring domains on CAS localization and dynamics in focal adhesions as well as on CAS-mediated mechanotransduction. We confirmed the predicted prerequisite of the focal adhesion targeting for CAS-dependent mechanosensing and unraveled the critical importance of CAS SH3 domain in mechanosensing. We further show that CAS localizes to the force transduction layer of focal adhesions and that mechanical stress stabilizes CAS in focal adhesions.

• MeSH
• buněčná adheze MeSH
• buněčný převod mechanických signálů * MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fokální adheze metabolismus   MeSH
• mechanický stres MeSH
• mutantní proteiny chemie   MeSH
• myši MeSH
• proteinové domény MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• signální transdukce MeSH
• stabilita proteinů MeSH
• substrátový protein asociovaný s Crk chemie   metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Bcar1 protein, mouse MeSH Prohlížeč
• mutantní proteiny MeSH
• rekombinantní fúzní proteiny MeSH
• substrátový protein asociovaný s Crk MeSH
• zelené fluorescenční proteiny MeSH

BACKGROUND: The local invasion of tumor cells into the surrounding tissue is the first and most critical step of the metastatic cascade. Cells can invade either collectively, or individually. Individual cancer cell invasion can occur in the mesenchymal or amoeboid mode, which are mutually interchangeable. This plasticity of individual cancer cell invasiveness may represent an escape mechanism for invading cancer cells from anti-metastatic treatment. METHODS: To identify new signaling proteins involved in the plasticity of cancer cell invasiveness, we performed proteomic analysis of the amoeboid to mesenchymal transition with A375m2 melanoma cells in a 3D Matrigel matrix. RESULTS: In this screen we identified PKCα as an important protein for the maintenance of amoeboid morphology. We found that the activation of PKCα resulted in the mesenchymal-amoeboid transition of mesenchymal K2 and MDA-MB-231 cell lines. Consistently, PKCα inhibition led to the amoeboid-mesenchymal transition of amoeboid A375m2 cells. Next, we showed that PKCα inhibition resulted in a considerable decrease in the invading abilities of all analyzed cancer cell lines. CONCLUSIONS: Our results suggest that PKCα is an important protein for maintenance of the amoeboid morphology of cancer cells, and that downregulation of PKCα results in the amoeboid to mesenchymal transition. Our data also suggest that PKCα is important for both mesenchymal and amoeboid invasiveness, making it an attractive target for anti-metastatic therapies.

• MeSH
• invazivní růst nádoru genetika   patologie   MeSH
• lidé MeSH
• melanom genetika   patologie   MeSH
• mezoderm metabolismus   patologie   MeSH
• nádorové buněčné linie MeSH
• pohyb buněk genetika   MeSH
• proteinkinasa C-alfa biosyntéza   genetika   MeSH
• proteomika MeSH
• regulace genové exprese u nádorů MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• PRKCA protein, human MeSH Prohlížeč
• proteinkinasa C-alfa MeSH

Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins, Crk-associated substrate (CAS) and vinculin, were both independently shown to be crucial for the ability of cells to transmit mechanical forces and to regulate cytoskeletal tension. Here, we identify a novel, direct binding interaction between CAS and vinculin. This interaction is mediated by the CAS SRC homology 3 domain and a proline-rich sequence in the hinge region of vinculin. We show that CAS localization in focal adhesions is partially dependent on vinculin, and that CAS-vinculin coupling is required for stretch-induced activation of CAS at the Y410 phosphorylation site. Moreover, CAS-vinculin binding significantly affects the dynamics of CAS and vinculin within focal adhesions as well as the size of focal adhesions. Finally, disruption of CAS binding to vinculin reduces cell stiffness and traction force generation. Taken together, these findings strongly implicate a crucial role of CAS-vinculin interaction in mechanosensing and focal adhesion dynamics.

• MeSH
• aminokyselinové motivy MeSH
• biomechanika MeSH
• buněčná adheze MeSH
• buněčné linie MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fokální adheze metabolismus   ultrastruktura   MeSH
• fokální adhezní tyrosinkinasy metabolismus   MeSH
• fosforylace MeSH
• mapy interakcí proteinů MeSH
• myši MeSH
• peptidy chemie   metabolismus   MeSH
• src homologní domény MeSH
• substrátový protein asociovaný s Crk analýza   metabolismus   MeSH
• vazba proteinů MeSH
• vinkulin analýza   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fokální adhezní tyrosinkinasy MeSH
• peptidy MeSH
• polyproline MeSH Prohlížeč
• substrátový protein asociovaný s Crk MeSH
• vinkulin MeSH

BACKGROUND: Although there is extensive evidence for the amoeboid invasiveness of cancer cells in vitro, much less is known about the role of amoeboid invasiveness in metastasis and the importance of Rho/ROCK/MLC signaling in this process. RESULTS: We analyzed the dependence of amoeboid invasiveness of rat and chicken sarcoma cells and the metastatic activity of chicken cells on individual elements of the Rho/ROCK/MLC pathway. In both animal models, inhibition of Rho, ROCK or MLC resulted in greatly decreased cell invasiveness in vitro, while inhibition of extracellular proteases using a broad spectrum inhibitor did not have a significant effect. The inhibition of both Rho activity and MLC phosphorylation by dominant negative mutants led to a decreased capability of chicken sarcoma cells to metastasize. Moreover, the overexpression of RhoA in non-metastatic chicken cells resulted in the rescue of both invasiveness and metastatic capability. Rho and ROCK, unlike MLC, appeared to be directly involved in the maintenance of the amoeboid phenotype, as their inhibition resulted in the amoeboid-mesenchymal transition in analyzed cell lines. CONCLUSION: Taken together, these results suggest that protease-independent invasion controlled by elements of the Rho/ROCK/MLC pathway can be frequently exploited by metastatic sarcoma cells.

• MeSH
• invazivní růst nádoru MeSH
• kinázy asociované s Rho metabolismus   MeSH
• krysa rodu Rattus MeSH
• kur domácí MeSH
• lehké řetězce myosinu metabolismus   MeSH
• nádorové buněčné linie MeSH
• pohyb buněk MeSH
• Rho proteiny vázající GTP metabolismus   MeSH
• sarkom metabolismus   patologie   MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kinázy asociované s Rho MeSH
• lehké řetězce myosinu MeSH
• Rho proteiny vázající GTP MeSH

UNLABELLED: Invasive cell growth and migration is usually considered a specifically metazoan phenomenon. However, common features and mechanisms of cytoskeletal rearrangements, membrane trafficking and signalling processes contribute to cellular invasiveness in organisms as diverse as metazoans and plants - two eukaryotic realms genealogically connected only through the last common eukaryotic ancestor (LECA). By comparing current understanding of cell invasiveness in model cell types of both metazoan and plant origin (invadopodia of transformed metazoan cells, neurites, pollen tubes and root hairs), we document that invasive cell behavior in both lineages depends on similar mechanisms. While some superficially analogous processes may have arisen independently by convergent evolution (e.g. secretion of substrate- or tissue-macerating enzymes by both animal and plant cells), at the heart of cell invasion is an evolutionarily conserved machinery of cellular polarization and oriented cell mobilization, involving the actin cytoskeleton and the secretory pathway. Its central components - small GTPases (in particular RHO, but also ARF and Rab), their specialized effectors, actin and associated proteins, the exocyst complex essential for polarized secretion, or components of the phospholipid- and redox- based signalling circuits (inositol-phospholipid kinases/PIP2, NADPH oxidases) are aparently homologous among plants and metazoans, indicating that they were present already in LECA. REVIEWER: This article was reviewed by Arcady Mushegian, Valerian Dolja and Purificacion Lopez-Garcia.

• MeSH
• aktiny metabolismus   MeSH
• cytoskelet metabolismus   MeSH
• lidé MeSH
• pohyb buněk fyziologie   MeSH
• pylová láčka cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• aktiny MeSH

BACKGROUND: SH3 domains are eukaryotic protein domains that participate in a plethora of cellular processes including signal transduction, proliferation, and cellular movement. Several studies indicate that tyrosine phosphorylation could play a significant role in the regulation of SH3 domains. RESULTS: To explore the incidence of the tyrosine phosphorylation within SH3 domains we queried the PhosphoSite Plus database of phosphorylation sites. Over 100 tyrosine phosphorylations occurring on 20 different SH3 domain positions were identified. The tyrosine corresponding to c-Src Tyr-90 was by far the most frequently identified SH3 domain phosphorylation site. A comparison of sequences around this tyrosine led to delineation of a preferred sequence motif ALYD(Y/F). This motif is present in about 15% of human SH3 domains and is structurally well conserved. We further observed that tyrosine phosphorylation is more abundant than serine or threonine phosphorylation within SH3 domains and other adaptor domains, such as SH2 or WW domains. Tyrosine phosphorylation could represent an important regulatory mechanism of adaptor domains. CONCLUSIONS: While tyrosine phosphorylation typically promotes signaling protein interactions via SH2 or PTB domains, its role in SH3 domains is the opposite - it blocks or prevents interactions. The regulatory function of tyrosine phosphorylation is most likely achieved by the phosphate moiety and its charge interfering with binding of polyproline helices of SH3 domain interacting partners.

• MeSH
• biologická evoluce * MeSH
• fosforylace MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• src homologní domény * MeSH
• tyrosin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• tyrosin MeSH