CMGC kinaasit ja syöpä

A decade after identification of the human kinome, only a fraction of the kinome has been systematically studied. This clearly illustrates the lack of knowledge regarding the functions and cellular effects that these proteins have in the establishment of cellular phenotypes. Surprisingly, recent evidence suggests that approximately 23% of all kinase genes were estimated to function as cancer genes. The overall aim of this thesis project was to shed light on the mechanisms controlling protein kinase activity and function, particularly in human cancers. A global and comprehensive interactomics analysis of the signaling networks of the evolutionarily conserved CMGC-protein kinase family, consisting of cyclin-dependent kinases [CDK], mitogen-activated protein kinases [MAPK], glycogen synthase kinases [GSK3] and CDC-like kinases [CLK]) was performed. From this physical and functional interaction analysis, hundreds of novel interactions, kinase-substrate relationships and previously unknown links to human diseases, such as cancer were identified. The unprecedented sensitivity and specificity of this approach was also illustrated, and highlighted, for example, by the purification and identification of the complete translational co-activator complex (the Mediator complex) with two of the CMGC kinases (CDK8 and CDK19). At the same time, the Mediator complex was identified mutated in benign uterine human tumors (uterine leiomyomas). To further deepen the understanding of the role of CDK8 and CDK19 kinases in controlling human disease pathways, we studied the functional role of two MED12 mutations in uterine leiomyoma and prostate cancer. Based on these studies, MED12 was found essential for the kinase activity of CDK8 in the context of the Mediator kinase module. Furthermore, disruption of the Mediator kinase module subunit interactions was shown as a common mechanism contributing to the formation of uterine leiomyoma. The MED12 mutants in leiomyoma and prostate cancer were shown functionally different. Finally, a novel MED12 nuclear localization signal was identified, and its importance in the correct nuclear localization of MED12 and subsequent proper assembly and function of the Mediator kinase module was experimentally proven. The described results from the multidisciplinary studies clarify the cellular roles of CMGC kinases and Mediator subunits, facilitating the design of targeted future therapies/therapeutics against human diseases.Millä tahansa ajanhetkellä, missä tahansa solussa, monet erilaiset molekyylien väliset signalointiverkostot ovat aktiivisia. Näiden verkostojen olennainen piirre on proteiinien fosforylaatioasteen säädeltävyys. Proteiinikinaasit välittävät useimpia solun signalointitapahtumia fosforyloimalla tietyn substraatin – muuttaen näiden aktiivisuutta, paikkaa solussa, ja/tai yhteyttä muihin proteiineihin. Proteiinifosfataasit puolestaan vastaavat defosforylaatiosta. Tällä hetkellä, vuosikymmen ihmisen kinomin annotaation jälkeen, suuri osa proteiinikinaasien ominaisuuksia selvittävistä tutkimuksista on yhä pienimuotoisia, yhteen kinaasiin keskittyviä. Tämän seurauksena proteiinikinaasien laaja-alainen tuntemus solun toimintojen säätelijöinä puuttuu. Yllättävää, sillä viimeaikaisen tutkimuksen valossa jopa 23% kinaasigeeneistä saattaa toimia syöpägeeneinä. Tämän tutkimuksen tarkoituksena oli muodostaa kokonaiskuva yhden keskeisimmän kinaasiperheen, CMGC-proteiinikinaasien, toiminnasta erityisesti syövässä. CMGC-kinaasiperheeseen kuuluu muun muassa CDK-, MAP-, GSK- sekä CLK-kinaaseja, joilla on keskeinen rooli solun toimintojen säätelyssä. Kattavasta proteiini-proteiini signalointiverkostojen analyysistämme tunnistimme satoja uusia proteiinien välisiä vuorovaikutuksia, kinaasi-substraatti pareja sekä aiemmin tuntemattomia yhteyksiä ihmisen sairauksiin, kuten syöpään. Pystyimme myös määrittämään kvantitatiivisesti kokonaisen translaatiokompleksin, Mediaattori-kompleksin, kaikki komponentit, mukaan lukien sen toimintaa säätelevän kinaasimoduulin. Samaan aikaan Mediaattori-kompleksin komponentin (MED12) mutaatioilla osoitettiin olevan rooli hyvänlaatuisissa ihmisen kohdun lihaskasvaimissa (leiomyooma). Ymmärtääksemme paremmin kahden Mediaattori-kompleksiin liittyvän CDK-kinaasin; CDK8 ja CDK19, roolia kasvainten kasvussa, tutkimme MED12 mutaatioiden vaikutusta kohdun lihaskasvaimissa sekä eturauhassyövässä. Näissä tutkimuksissa havaitsimme, että MED12:n toiminta on olennaista, sillä kohdun lihaskasvaimessa tapahtunut mutaatio esti kahden CMGC-kinaasin, CDK8 ja CDK19, vuorovaikutuksen kohdeproteiininsa kanssa aiheuttaen siten kasvaimen kasvun. Lisäksi havaitsimme, että MED12 mutaatiot kohdun lihaskasvaimissa ja eturauhassyövässä ovat toiminnallisesti erilaisia. Paikallistimme aiemmin tuntemattoman tumalokalisaatiosignaalin MED12-proteiinissa, ja pystyimme osoittamaan sen tärkeyden kyseisen proteiinin toiminnalle. Tämä väitöstutkimus on esimerkki monialaisesta tutkimusyhteistyöstä, jonka tuloksena selvitettiin kasvainbiologiaa ja signaaliverkostoja MED12-proteiinin ja CMGC-kinaasiperheen osalta. Solun signalointireittien toiminnan yksityiskohtainen tunteminen on hyvin keskeistä syövän syntymekanismien ymmärtämiseksi sekä uusien ja kohdennettujen lääkehoitojen kehittämiseksi

Altered glycosylation of several metastasis-associated glycoproteins with terminal GalNAc defines the highly invasive cancer cell phenotype

Publisher Copyright: © 2022 Khosrowabadi et al.Several distinct metastasis-associated glycosylation changes have been shown to promote cancer cell invasion and metastasis, the main cause of death of cancer patients. However, it is unclear whether their presence reflects cell- or tissue-specific variations for metastasis, or species needed to drive different phases of the metastatic cascade. To address this issue from a different perspective, we investigated here whether different cancer cell lines share any glycotopes that are common and important for their invasive phenotype. By using lectin microarray glycan profiling and an established myoma tissue-based 3D invasion assay, we identified a single glycotope recognized by Helix Pomatia agglutinin (HPA), whose expression level in different cancer cells correlated significantly with their invasive potential. Lectin pull-down assay and LC-MS/MS analysis in highly- (A431 and SW-48) and poorly invasive (HepG2 and RCC4) cancer cells revealed ~85 glycoproteins of which several metastasis-promoting members of the integrin family of cell adhesion receptors, the epidermal growth factor receptor (EGFR) and the matrix metalloproteinase-14 (MMP-14) were among the abundant ones. Moreover, we showed that the level of the GalNAc glycotope in MMP-14, EGFR, αV-, β1- and β4 integrin in highly and poorly invasive cancer cells correlated positively with their invasive potential. Collectively, our findings suggest that altered glycosylation of several metastasis-associated glycoproteins with terminal GalNAc drives the highly invasive cancer cell phenotype.Peer reviewe

Physical and functional interactome atlas of human receptor tyrosine kinases

Funding Information: We thank S. Miettinen for technical assistance and Professors Matthias Gstaiger, Aki Manninen, and Kaisa Lehti for critical reading and comments on the manuscript. This study was supported by grants from the Academy of Finland (nos. 288475 and 294173), the Sigrid Jusélius Foundation, the Finnish Cancer Foundation, the University of Helsinki Three‐year Research Grant, Biocentrum Helsinki, Biocentrum Finland, HiLIFE, Magnus Ehrnrooth Foundation, and the Instrumentarium Research Foundation. Open access funding enabled and organized by ProjektDEAL. Publisher Copyright: © 2022 The Authors. Published under the terms of the CC BY 4.0 license.Much cell-to-cell communication is facilitated by cell surface receptor tyrosine kinases (RTKs). These proteins phosphorylate their downstream cytoplasmic substrates in response to stimuli such as growth factors. Despite their central roles, the functions of many RTKs are still poorly understood. To resolve the lack of systematic knowledge, we apply three complementary methods to map the molecular context and substrate profiles of RTKs. We use affinity purification coupled to mass spectrometry (AP-MS) to characterize stable binding partners and RTK–protein complexes, proximity-dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates. To identify how kinase interactions depend on kinase activity, we also use kinase-deficient mutants. Our data represent a comprehensive, systemic mapping of RTK interactions and substrates. This resource adds information regarding well-studied RTKs, offers insights into the functions of less well-studied RTKs, and highlights RTK-RTK interactions and shared signaling pathways.Peer reviewe

IRF2BP2 Mutation Is Associated with Increased STAT1 and STAT5 Activation in Two Family Members with Inflammatory Conditions and Lymphopenia

Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a transcriptional coregulator that has an important role in the regulation of the immune response. IRF2BP2 has been associated with the Janus kinase (JAK)—signal transducers and activators of transcription (STAT) pathway, but its exact role remains elusive. Here, we identified a novel clinical variant, IRF2BP2 c.625_665del, from two members of a family with inflammatory conditions and investigated the function of IRF2BP2 and c.625_665del mutation in JAK–STAT pathway activation and inflammatory signaling. The levels of constitutive and cytokine-induced phosphorylation of STATs and total STAT1 in peripheral blood monocytes, T cells, and B cells from the patients and four healthy controls were measured by flow cytometry. Inflammation-related gene expression was studied in peripheral blood mononuclear cells using direct digital detection of mRNA (NanoString). Finally, we studied the relationship between IRF2BP2 and STAT1 activation using a luciferase reporter system in a cell model. Our results show that patients having the IRF2BP2 c.625_665del mutation presented overexpression of STAT1 protein and increased constitutive activation of STAT1. In addition, interferon-induced JAK–STAT signaling was upregulated, and several interferon-inducible genes were overexpressed. Constitutive phosphorylation of STAT5 was also found to be upregulated in CD4+ T cells from the patients. Using a cell model, we show that IRF2BP2 was needed to attenuate STAT1 transcriptional activity and that IRF2BP2 c.625_665del mutation failed in this. We conclude that IRF2BP2 has an important role in suppressing immune responses elicited by STAT1 and STAT5 and suggest that aberrations in IRF2BP2 can lead to abnormal function of intrinsic immunity

IRF2BP2 Mutation Is Associated with Increased STAT1 and STAT5 Activation in Two Family Members with Inflammatory Conditions and Lymphopenia

Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a transcriptional coregulator that has an important role in the regulation of the immune response. IRF2BP2 has been associated with the Janus kinase (JAK)—signal transducers and activators of transcription (STAT) pathway, but its exact role remains elusive. Here, we identified a novel clinical variant, IRF2BP2 c.625_665del, from two members of a family with inflammatory conditions and investigated the function of IRF2BP2 and c.625_665del mutation in JAK–STAT pathway activation and inflammatory signaling. The levels of constitutive and cytokine-induced phosphorylation of STATs and total STAT1 in peripheral blood monocytes, T cells, and B cells from the patients and four healthy controls were measured by flow cytometry. Inflammation-related gene expression was studied in peripheral blood mononuclear cells using direct digital detection of mRNA (NanoString). Finally, we studied the relationship between IRF2BP2 and STAT1 activation using a luciferase reporter system in a cell model. Our results show that patients having the IRF2BP2 c.625_665del mutation presented overexpression of STAT1 protein and increased constitutive activation of STAT1. In addition, interferon-induced JAK–STAT signaling was upregulated, and several interferon-inducible genes were overexpressed. Constitutive phosphorylation of STAT5 was also found to be upregulated in CD4+ T cells from the patients. Using a cell model, we show that IRF2BP2 was needed to attenuate STAT1 transcriptional activity and that IRF2BP2 c.625_665del mutation failed in this. We conclude that IRF2BP2 has an important role in suppressing immune responses elicited by STAT1 and STAT5 and suggest that aberrations in IRF2BP2 can lead to abnormal function of intrinsic immunity

The Protein Interaction Landscape of the Human CMGC Kinase Group

Peer reviewe

Haploinsufficiency of A20 impairs protein–protein interactome and leads into caspase-8-dependent enhancement of NLRP3 inflammasome activation

Objectives TNFAIP3 encodes A20 that negatively regulates nuclear factor kappa light chain enhancer of activated B cells (NF-κB), the major transcription factor coordinating inflammatory gene expression. TNFAIP3 polymorphisms have been linked with a spectrum of inflammatory and autoimmune diseases and, recently, loss-of-function mutations in A20 were found to cause a novel inflammatory disease ‘haploinsufficiency of A20’ (HA20). Here we describe a family with HA20 caused by a novel TNFAIP3 loss-of-function mutation and elucidate the upstream molecular mechanisms linking HA20 to dysregulation of NF-κB and the related inflammasome pathway.Methods NF-κB activation was studied in a mutation-expressing cell line using luciferase reporter assay. Physical and close-proximity protein–protein interactions of wild-type and TNFAIP3 p.(Lys91*) mutant A20 were analysed using mass spectrometry. NF-κB -dependent transcription, cytokine secretion and inflammasome activation were compared in immune cells of the HA20 patients and control subjects.Results The protein–protein interactome of p.(Lys91*) mutant A20 was severely impaired, including interactions with proteins regulating NF-κB activation, DNA repair responses and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. The p.(Lys91*) mutant A20 failed to suppress NF-κB signalling, which led to increased NF-κB -dependent proinflammatory cytokine transcription. Functional experiments in the HA20 patients’ immune cells uncovered a novel caspase-8-dependent mechanism of NLRP3 inflammasome hyperresponsiveness that mediated the excessive secretion of interleukin-1β and interleukin-18.Conclusions The current findings significantly deepen our understanding of the molecular mechanisms underlying HA20 and other diseases associated with reduced A20 expression or function, paving the way for future therapeutic targeting of the pathway.Peer reviewe

Modified VEGF-A with improved angiogenic properties

The present invention is directed to methods and compositions for making and using chimeric polypeptides that comprise a VEGFR-2 ligand. The chimeric molecules of the present invention retain VEGFR-2 binding activity and an enhanced angiogenic activity as compared to native VEGF-A

Combined immunodeficiency and hypoglycemia associated with mutations in hypoxia upregulated 1

Non peer reviewe

Characterization of Expanded Gamma Delta T Cells from Atypical X-SCID Patient Reveals Preserved Function and IL2RG-Mediated Signaling

Abnormally high gamma delta T cell numbers among individuals with atypical SCID have been reported but detailed immunopheno typing and functional characterization of these expanded gamma delta T cells are limited. We have previously reported atypical SCID phenotype caused by hypomorphic IL2RG (NM_000206.3) c.172C > T;p.(Pro58Ser) variant. Here, we have further investigated the index patient's abnormally large gamma delta T cell population in terms of function and phenotype by studying IL2RG cell surface expression, STAT tyrosine phosphorylation and blast formation in response to interleukin stimulation, immunophenotyping, TCRv gamma sequencing, and target cell killing. In contrast to his alpha beta T cells, the patient's gamma delta T cells showed normal IL2RG cell surface expression and normal or enhanced IL2RG-mediated signaling. V delta 2 + population was proportionally increased with a preponderance of memory phenotypes and high overall tendency towards perforin expression. The patient's gamma delta T cells showed enhanced cytotoxicity towards A549 cancer cells. His TCRv gamma repertoire was versatile but sequencing of IL2RG revealed a novel c.534C > A; p.(Phe178Leu) somatic missense variant restricted to gamma delta T cells. Over time this variant became predominant in gamma delta T cells, though initially present only in part of them. IL2RG-Pro58Ser/Phe178Leu variant showed higher cell surface expression compared to IL2RG-Pro58Ser variant in stable HEK293 cell lines, suggesting that somatic p.(Phe178Leu) variant may at least partially rescue the pathogenic effect of germline p.(Pro58Ser) variant. In conclusion, our report indicates that expansion of gamma delta T cells associated with atypical SCID needs further studying and cannot exclusively be deemed as a homeostatic response to low numbers of conventional T cells.Peer reviewe