Calpain2 is a conventional member of the non-lysosomal calpain protease family that has been shown to affect the dynamics of focal and cell-cell adhesions by proteolyzing the components of adhesion complexes. Here, we inactivated calpain2 using CRISPR/Cas9 in epithelial MDCK cells. We show that depletion of calpain2 has multiple effects on cell morphology and function. Calpain2-depleted cells develop epithelial shape, however, they cover a smaller area, and cell clusters are more compact. Inactivation of calpain2 enhanced restoration of transepithelial electrical resistance after calcium switch, decreased cell migration, and delayed cell scattering induced by HGF/SF. In addition, calpain2 depletion prevented morphological changes induced by ERK2 overexpression. Interestingly, proteolysis of several calpain2 targets, including E-cadherin, β-catenin, talin, FAK, and paxillin, was not discernibly affected by calpain2 depletion. Taken together, these data suggest that calpain2 regulates the stability of cell-cell and cell-substratum adhesions indirectly without affecting the proteolysis of these adhesion complexes.

• Klíčová slova
• Actin, Adherens junctions, Calpains, Cell scattering, ERK, Epithelial polarity, Focal adhesions, HGF/SF, Migration, Proteases, Tight junctions, Transepithelial electrical resistance,
• MeSH
• beta-katenin metabolismus   MeSH
• buněčná adheze * MeSH
• buňky MDCK MeSH
• CRISPR-Cas systémy MeSH
• epitelové buňky * metabolismus   cytologie   MeSH
• hepatocytární růstový faktor metabolismus   MeSH
• kadheriny metabolismus   MeSH
• kalpain * metabolismus   MeSH
• mitogenem aktivovaná proteinkinasa 1 metabolismus   MeSH
• pohyb buněk MeSH
• proteolýza MeSH
• psi MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-katenin MeSH
• hepatocytární růstový faktor MeSH
• kadheriny MeSH
• kalpain * MeSH
• mitogenem aktivovaná proteinkinasa 1 MeSH
• vápník MeSH

• Publikační typ
• tisková chyba MeSH

The ERK signaling pathway, consisting of core protein kinases Raf, MEK and effector kinases ERK1/2, regulates various biological outcomes such as cell proliferation, differentiation, apoptosis, or cell migration. Signal transduction through the ERK signaling pathway is tightly controlled at all levels of the pathway. However, it is not well understood whether ERK pathway signaling can be modulated by the abundance of ERK pathway core kinases. In this study, we investigated the effects of low-level overexpression of the ERK2 isoform on the phenotype and scattering of cuboidal MDCK epithelial cells growing in discrete multicellular clusters. We show that ERK2 overexpression reduced the vertical size of lateral membranes that contain cell-cell adhesion complexes. Consequently, ERK2 overexpressing cells were unable to develop cuboidal shape, remained flat with increased spread area and intercellular adhesive contacts were present only on the basal side. Interestingly, ERK2 overexpression was not sufficient to increase phosphorylation of multiple downstream targets including transcription factors and induce global changes in gene expression, namely to increase the expression of pro-migratory transcription factor Fra1. However, ERK2 overexpression enhanced HGF/SF-induced cell scattering as these cells scattered more rapidly and to a greater extent than parental cells. Our results suggest that an increase in ERK2 expression primarily reduces cell-cell cohesion and that weakened intercellular adhesion synergizes with upstream signaling in the conversion of the multicellular epithelium into single migrating cells. This mechanism may be clinically relevant as the analysis of clinical data revealed that in one type of cancer, pancreatic adenocarcinoma, ERK2 overexpression correlates with a worse prognosis.

• Klíčová slova
• Cell scattering, Cell-cell adhesions, ERK, Epithelial plasticity, Fra1, HGF/SF,
• MeSH
• adenokarcinom * metabolismus   MeSH
• buněčná adheze MeSH
• epitelové buňky metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• MAP kinasový signální systém fyziologie   MeSH
• mitogenem aktivovaná proteinkinasa 1 MeSH
• mitogenem aktivované proteinkinasy kinas metabolismus   MeSH
• nádory slinivky břišní * metabolismus   MeSH
• proliferace buněk MeSH
• signální transdukce MeSH
• transkripční faktory metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• MAPK1 protein, human MeSH Prohlížeč
• mitogenem aktivovaná proteinkinasa 1 MeSH
• mitogenem aktivované proteinkinasy kinas MeSH
• transkripční faktory MeSH

β-Arrestins are known to play a crucial role in GPCR-mediated transmembrane signaling processes. However, there are still many unanswered questions, especially those concerning the presumed similarities and differences of β-arrestin isoforms. Here, we examined the roles of β-arrestin 1 and β-arrestin 2 at different levels of μ-opioid receptor (MOR)-regulated signaling, including MOR mobility, internalization of MORs, and adenylyl cyclase (AC) activity. For this purpose, naïve HEK293 cells or HEK293 cells stably expressing YFP-tagged MOR were transfected with appropriate siRNAs to block in a specific way the expression of β-arrestin 1 or β-arrestin 2. We did not find any significant differences in the ability of β-arrestin isoforms to influence the lateral mobility of MORs in the plasma membrane. Using FRAP and line-scan FCS, we observed that knockdown of both β-arrestins similarly increased MOR lateral mobility and diminished the ability of DAMGO and endomorphin-2, respectively, to enhance and slow down receptor diffusion kinetics. However, β-arrestin 1 and β-arrestin 2 diversely affected the process of agonist-induced MOR endocytosis and exhibited distinct modulatory effects on AC function. Knockdown of β-arrestin 1, in contrast to β-arrestin 2, more effectively suppressed forskolin-stimulated AC activity and prevented the ability of activated-MORs to inhibit the enzyme activity. Moreover, we have demonstrated for the first time that β-arrestin 1, and partially β-arrestin 2, may somehow interact with AC and that this interaction is strongly supported by the enzyme activation. These data provide new insights into the functioning of β-arrestin isoforms and their distinct roles in GPCR-mediated signaling.

• Klíčová slova
• Adenylyl cyclase, DAMGO, Endomorphin-2, Receptor lateral mobility, β-Arrestin, μ-Opioid receptor,
• MeSH
• adenylátcyklasy * metabolismus   MeSH
• beta arrestin 1 metabolismus   MeSH
• beta arrestin 2 metabolismus   MeSH
• beta arrestiny metabolismus   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• receptory opiátové mu * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasy * MeSH
• ARRB1 protein, human MeSH Prohlížeč
• beta arrestin 1 MeSH
• beta arrestin 2 MeSH
• beta arrestiny MeSH
• receptory opiátové mu * MeSH

Dishevelled (DVL) proteins are key mediators of most Wnt pathways. In all vertebrates, three DVL paralogs are present (DVL1, DVL2 and DVL3) but it is poorly defined to what extent they are functionally redundant. Here, we generated T-REx HEK 293 cells with only one DVL paralog (i.e., DVL1-only, DVL2-only, and DVL3-only) and compared their response to Wnt-3a and Wnt-5a ligands with wild type and DVL triple knockout cells. We show that DVL is essential, in addition to the previously shown Wnt-3a-induced phosphorylation of LRP6 and transcriptional activation of TCF/LEF-dependent reporter, also for Wnt-3a-induced degradation of AXIN1 and Wnt-5a-induced phosphorylation of ROR1. We have quantified the molar ratios of DVL1:DVL2:DVL3 in our model to be approximately 4:80:16. Interestingly, DVL-only cells do not compensate for the lack of other paralogs and are still fully functional in all analyzed readouts with the exception of Wnt-3a-induced transcription assessed by TopFlash assay. In this assay, the DVL1-only cell line was the most potent; on the contrary, the DVL3-only cell line exhibited only the negligible capacity to mediate Wnt signals. Using a novel model system - complementation assays in T-REx HEK 293 with amplified Wnt signal response (RNF43/ZNRF3/DVL1/DVL2/DVL3 penta KO cells) we demonstrate that it is not the total amount of DVL but ratio of individual paralogs what decides the signal strength. In sum, this study contributes to our better understanding of the role of individual human DVL paralogs in the Wnt pathway.

• Klíčová slova
• Axin1, CRISPR/Cas9, Dishevelled 1/2/3, ROR1, Wnt signalling,
• MeSH
• aktivace transkripce MeSH
• fosfoproteiny * metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• lidé MeSH
• protein dishevelled metabolismus   MeSH
• signální dráha Wnt * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoproteiny * MeSH
• protein dishevelled MeSH

Interferon-induced transmembrane proteins IFITM1 and IFITM3 (IFITM1/3) play a role in both RNA viral restriction and in human cancer progression. Using immunohistochemical staining of FFPE tissue, we identified subgroups of cervical cancer patients where IFITM1/3 protein expression is inversely related to metastasis. Guide RNA-CAS9 methods were used to develop an isogenic IFITM1/IFITM3 double null cervical cancer model in order to define dominant pathways triggered by presence or absence of IFITM1/3 signalling. A pulse SILAC methodology identified IRF1, HLA-B, and ISG15 as the most dominating IFNγ inducible proteins whose synthesis was attenuated in the IFITM1/IFITM3 double-null cells. Conversely, SWATH-IP mass spectrometry of ectopically expressed SBP-tagged IFITM1 identified ISG15 and HLA-B as dominant co-associated proteins. ISG15ylation was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. Proximity ligation assays indicated that HLA-B can interact with IFITM1/3 proteins in parental SiHa cells. Cell surface expression of HLA-B was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. SWATH-MS proteomic screens in cells treated with IFITM1-targeted siRNA cells resulted in the attenuation of an interferon regulated protein subpopulation including MHC Class I molecules as well as IFITM3, STAT1, B2M, and ISG15. These data have implications for the function of IFITM1/3 in mediating IFNγ stimulated protein synthesis including ISG15ylation and MHC Class I production in cancer cells. The data together suggest that pro-metastatic growth associated with IFITM1/3 negative cervical cancers relates to attenuated expression of MHC Class I molecules that would support tumor immune escape.

• Klíčová slova
• CAS9 gene editing, Cervical cancer, IFITM1, Interferon, MHC Class I molecule, SILAC mass spectrometry,
• MeSH
• buněčné linie MeSH
• diferenciační antigeny fyziologie   MeSH
• histokompatibilita - antigeny třídy I metabolismus   MeSH
• lidé MeSH
• membránové proteiny fyziologie   MeSH
• nádory děložního čípku metabolismus   MeSH
• proteiny vázající RNA fyziologie   MeSH
• proteosyntéza fyziologie   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diferenciační antigeny MeSH
• histokompatibilita - antigeny třídy I MeSH
• IFITM3 protein, human MeSH Prohlížeč
• leu-13 antigen MeSH Prohlížeč
• membránové proteiny MeSH
• proteiny vázající RNA MeSH

AIM: Heat shock protein 60 (Hsp60) is a mediator of stress-induced vascular smooth muscle cell (VSMC) proliferation. This study will determine, first, if the mitochondrial or cytoplasmic localization of Hsp60 is critical to VSMC proliferation and, second, the mechanism of Hsp60 induction of VSMC proliferation with a focus on modification of nucleocytoplasmic trafficking. METHODS AND RESULTS: Hsp60 was overexpressed in primary rabbit VSMCs with or without a mitochondrial targeting sequence (AdHsp60mito-). Both interventions induced an increase in VSMC PCNA expression and proliferation. The increase in VSMC PCNA expression and growth was not observed after siRNA-mediated knockdown of Hsp60 expression. Nuclear protein import in VSMC was measured by fluorescent microscopy using a microinjected fluorescent import substrate. Nuclear protein import was stimulated by both AdHsp60 and AdHsp60mito- treatments. AdHsp60 treatment also induced increases in nucleoporin (Nup) 62, Nup153, importin-α, importin-β and Ran expression as well as cellular ATP levels compared to control. AdHsp60mito- treatment induced an up-regulation in importin-α, importin-β and Ran expression compared to control. Hsp60 knockdown did not change nuclear protein import nor the expression of any nuclear transport receptors or nucleoporins. Both heat shock treatment and Hsp60 overexpression promoted the interaction of Ran with Hsp60. CONCLUSIONS: VSMC proliferation can be modulated via an Hsp60 dependent, cytosol localized mechanism that in part involves a stimulation of nuclear protein import through an interaction with Ran. This novel cellular signaling role for Hsp60 may be important in growth-based vascular pathologies like atherosclerosis and hypertension.

• Klíčová slova
• Cell proliferation *, Heat shock protein 60 *, Nuclear protein import *, Nucleocytoplasmic trafficking *, Smooth muscle *,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• chaperon hsp60 antagonisté a inhibitory   genetika   metabolismus   MeSH
• komplex proteinů jaderného póru metabolismus   MeSH
• králíci MeSH
• kultivované buňky MeSH
• malá interferující RNA metabolismus   MeSH
• proliferace buněk * MeSH
• Ran protein vázající GTP genetika   metabolismus   MeSH
• RNA interference MeSH
• svaly hladké cévní cytologie   metabolismus   MeSH
• teplota MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• chaperon hsp60 MeSH
• komplex proteinů jaderného póru MeSH
• malá interferující RNA MeSH
• Ran protein vázající GTP MeSH

Receptor tyrosine kinases (RTKs) form multiprotein complexes that initiate and propagate intracellular signals and determine the RTK-specific signalling patterns. Unravelling the full complexity of protein interactions within the RTK-associated complexes is essential for understanding of RTK functions, yet it remains an understudied area of cell biology. We describe a comprehensive approach to characterize RTK interactome. A single tag immunoprecipitation and phosphotyrosine protein isolation followed by mass-spectrometry was used to identify proteins interacting with fibroblast growth factor receptor 3 (FGFR3). A total of 32 experiments were carried out in two different cell types and identified 66 proteins out of which only 20 (30.3%) proteins were already known FGFR interactors. Using co-immunoprecipitations, we validated FGFR3 interaction with adapter protein STAM1, transcriptional regulator SHOX2, translation elongation factor eEF1A1, serine/threonine kinases ICK, MAK and CCRK, and inositol phosphatase SHIP2. We show that unappreciated signalling mediators exist for well-studied RTKs, such as FGFR3, and may be identified via proteomic approaches described here. These approaches are easily adaptable to other RTKs, enabling identification of novel signalling mediators for majority of the known human RTKs.

• Klíčová slova
• FGFR3, Fibroblast growth factor, interactome, receptor tyrosine kinase, signal transduction,
• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• buňky NIH 3T3 MeSH
• cyklin-dependentní kinasy genetika   metabolismus   MeSH
• elongační faktor 1 genetika   metabolismus   MeSH
• endozomální třídící komplexy pro transport genetika   metabolismus   MeSH
• fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy genetika   metabolismus   MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• kinasa aktivující cyklin dependentní kinasy MeSH
• lidé MeSH
• mapování interakce mezi proteiny MeSH
• myši MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• proteomika metody   MeSH
• receptor fibroblastových růstových faktorů, typ 3 genetika   metabolismus   MeSH
• regulace genové exprese * MeSH
• signální transdukce genetika   MeSH
• stanovení celkové genové exprese MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• CILK1 protein, human MeSH Prohlížeč
• cyklin-dependentní kinasy MeSH
• EEF1A1 protein, human MeSH Prohlížeč
• elongační faktor 1 MeSH
• endozomální třídící komplexy pro transport MeSH
• FGFR3 protein, human MeSH Prohlížeč
• fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy MeSH
• fosfoproteiny MeSH
• homeodoménové proteiny MeSH
• INPPL1 protein, human MeSH Prohlížeč
• kinasa aktivující cyklin dependentní kinasy MeSH
• MAK protein, human MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• receptor fibroblastových růstových faktorů, typ 3 MeSH
• SHOX2 protein, human MeSH Prohlížeč
• STAM protein, human MeSH Prohlížeč

Frizzleds (FZDs) are unconventional G protein-coupled receptors, which activate diverse intracellular signaling pathways via the phosphoprotein Disheveled (DVL) and heterotrimeric G proteins. The interaction interplay of FZDs with DVL and G proteins is complex, involves different regions of FZD and the potential dynamics are poorly understood. In the present study, we aimed to characterize the function of a highly conserved tyrosine (Y2502.39) in the intracellular loop 1 (IL1) of human FZD4. We have found Y2502.39 to be crucial for DVL2 interaction and DVL2 translocation to the plasma membrane. Mutant FZD4-Y2502.39F, impaired in DVL2 binding, was defective in both β-catenin-dependent and β-catenin-independent WNT signaling induced in Xenopus laevis embryos. The same mutant maintained interaction with the heterotrimeric G proteins Gα12 and Gα13 and was able to mediate WNT-induced G protein dissociation and G protein-dependent YAP/TAZ signaling. We conclude from modeling and dynamics simulation efforts that Y2502.39 is important for the structural integrity of the FZD-DVL, but not for the FZD-G protein interface and hypothesize that the interaction network of Y2502.39 and H3484.46 plays a role in specifying downstream signaling pathways induced by the receptor.

• Klíčová slova
• DVL2, Disheveled, FZD(4), Frizzled, GNA12, GNA13,
• MeSH
• aminokyselinové motivy MeSH
• embryo nesavčí metabolismus   MeSH
• frizzled receptory chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• heterotrimerní G-proteiny metabolismus   MeSH
• konzervovaná sekvence * MeSH
• lidé MeSH
• mutační analýza DNA MeSH
• nádory metabolismus   patologie   MeSH
• polymerizace MeSH
• protein dishevelled chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální dráha Wnt MeSH
• signální transdukce MeSH
• simulace molekulární dynamiky MeSH
• strukturní homologie proteinů MeSH
• tyrosin metabolismus   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Xenopus laevis embryologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• frizzled receptory MeSH
• heterotrimerní G-proteiny MeSH
• protein dishevelled MeSH
• tyrosin MeSH

Among the 10 Frizzled (FZD) isoforms belonging to the Class F of G protein-coupled receptors (GPCRs), FZD10 remains the most enigmatic. FZD10 shows homology to FZD4 and FZD9 and was previously implicated in both β-catenin-dependent and -independent signalling. In normal tissue, FZD10 levels are generally very low; however, its upregulation in synovial carcinoma has attracted some attention for therapy. Our findings identify FZD10 as a receptor interacting with and signalling through the heterotrimeric G protein Gα13 but not Gα12, Gαi1, GαoA, Gαs, or Gαq. Stimulation with the FZD agonist WNT induced the dissociation of the Gα13 protein from FZD10, and led to global Gα12/13-dependent cell changes assessed by dynamic mass redistribution measurements. Furthermore, we show that FZD10 mediates Gα12/13 activation-dependent induction of YAP/TAZ transcriptional activity. In addition, we show a distinct expression of FZD10 in embryonic CNS endothelial cells at E11.5-E14.5. Given the well-known importance of Gα13 signalling for the development of the vascular system, the selective expression of FZD10 in brain vascular endothelial cells points at a potential role of FZD10-Gα13 signalling in CNS angiogenesis.

• Klíčová slova
• Angiogenesis, Endothelial cell, FZD, GNA13, GPCR, WNT, YAP/TAZ,
• MeSH
• buněčné linie MeSH
• centrální nervový systém krevní zásobení   MeSH
• cévní endotel účinky léků   metabolismus   MeSH
• frizzled receptory metabolismus   MeSH
• fyziologická neovaskularizace * MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• protein dishevelled metabolismus   MeSH
• proteiny vázající GTP - alfa-podjednotky G12-G13 metabolismus   MeSH
• proteiny Wnt farmakologie   MeSH
• signální transdukce * MeSH
• vazba proteinů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• frizzled receptory MeSH
• FZD10 protein, human MeSH Prohlížeč
• Fzd10 protein, mouse MeSH Prohlížeč
• protein dishevelled MeSH
• proteiny vázající GTP - alfa-podjednotky G12-G13 MeSH
• proteiny Wnt MeSH