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Protein tyrosine kinases and the regulation of signalling and adhesion in drosophila melanogaster
Umeå universitet, Medicinsk fakultet, Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten).
2007 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

In order to build a multi-cellular organism and to regulate cellular functions, cells need to communicate with each other, as well as tightly regulate their behaviour in response to environmental changes. For these purposes all eukaryotic cells express a large number of membrane spanning receptors that either themselves contain catalytic activity or via cytoplasmic effector enzymes, function to transmit “signals” from the cell exterior to induce appropriate responses within the cell. Protein tyrosine kinases (PTKs) are important signalling molecules, represented by the transmembrane receptor tyrosine kinases (RTKs) in addition to the cytoplasmic non-receptor PTKs, which alter cell behaviour by phosphorylating target proteins. An additional requirement for proper signalling and multicellular organisation is the adhesion between cells as well as adhesion of cells to the extracellular matrix (ECM).

Adhesion between cells and the ECM is mainly mediated by the integrin family of cell surface receptors, which functions as a structural link between the ECM and the actin cytoskeleton as well as important centres for signalling. Mammalian studies have implicated the cytoplasmic Focal Adhesion Kinase (FAK), as a major transmitter of signalling emanating from integrins, regulating cell migration, survival, proliferation and differentiation. In our studies of the sole FAK family member in Drosophila, Fak56, we have concluded that the deletion of Fak56 from the fruit fly genome causes no obvious defects in integrin-mediated adhesion, migration or signalling in vivo. Consequently, in contrast to the embryonic lethality observed in mouse knockouts, Fak56 mutant flies are both viable and fertile. However, we do find a clear genetic interaction between Fak56 and Drosophila integrins. Additionally, overexpression studies indeed indicate Fak56 as a negative regulator of integrin adhesion, given that excess Fak56 protein phenocopies loss of integrin function, causing phenotypes such as muscle detachment and wing blistering.

In Drosophila, as well as in mammals, FAK family proteins are highly abundant in the CNS and in our studies we have identified a requirement of Fak56 in synaptic transmission at neuromuscular junctions. Lack of Fak56 causes a weakening of action potential conduction, resulting in sensitivity to high-frequency mechanical and electrical stimulation, manifested by epileptic-like seizures and paralysis in Fak56 mutants, a phenotype known as Bang Sensitivity (BS) in flies. We also show that Fak56 phosphorylation is directly modulated in response to alterations in intracellular calcium levels, supporting a role for Fak56 in neurotransmission.

Fak56 is directly activated by the Drosophila Anaplastic Lymphoma Kinase, DAlk, receptor which was identified in our lab. We characterised DAlk as a novel RTK that is expressed in the embryonic CNS and mesoderm where it drives activation of the ERK/MAPK pathway. Indeed, we found DAlk to ectopically induce protein tyrosine phosphorylation and specifically phosphorylation of ERK, resulting in autonomous cell transformation and uncontrolled tissue growth. Subsequently, we identified a requirement for DAlk function during Drosophila embryogenesis, where it displays an essential role in gut development. Specifically, we identified the secreted molecule Jelly belly (Jeb) as a ligand for DAlk and showed that Jeb-DAlk interaction activates an ERK-mediated signalling pathway essential for visceral muscle specification and fusion, and consequently formation of the gut.

The potent ability of PTKs to regulate cell behaviour, together with the strong linkage between RTK dysregulation and tumour formation, renders the negative regulation of kinase activity an important area of research. We have identified the Drosophila homologue of Cbl-interacting protein of 85kDa, dCIN85, an adaptor molecule which in mammalian cells has shown involvement in RTK endocytosis and downregulation, as well as in the regulation of actin cytoskeleton dynamics. In the fruit fly, dCIN85 displays essential functions, given that dCIN85 loss of function mutants display a grand-child less phenotype. Generation of a dCIN85 antibody, together with isoform-specific transgenic flies, have allowed us to observe a punctuate localization pattern of the SH3-domain containing dCIN85 variants, representing Rab5-positive endosomal structures. This, in addition to the confirmation of a direct dCIN85-dCbl interaction, indicates an evolutionary conservation of dCIN85 function. Interestingly, dCIN85 co-localises with dRICH1, a Cdc42 specific RhoGAP, in differentiated photoreceptor cells in eye imaginal discs. This may imply a role for dCIN85 in the regulation of the specialised endocytic recycling processes required for the assembly/maintenance of tight junctions and establishment of cell polarity in epithelial tissues.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå centrum för molekylär patogenes (UCMP) (Medicinska fakulteten) , 2007. , s. 104
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1080
Nyckelord [en]
Drosophila, Signalling, Adhesion, Protein Tyrosine Kinase, Development, Endocytosis
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-971ISBN: 978-91-7264-246-1 (tryckt)OAI: oai:DiVA.org:umu-971DiVA, id: diva2:145247
Disputation
2007-02-09, Hörsal E04, 6L, Norrlands Universitetssjukhus, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2007-01-19 Skapad: 2007-01-19 Senast uppdaterad: 2009-05-19Bibliografiskt granskad
Delarbeten
1. Focal adhesion kinase is not required for integrin function or viability in Drosophila.
Öppna denna publikation i ny flik eller fönster >>Focal adhesion kinase is not required for integrin function or viability in Drosophila.
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2004 (Engelska)Ingår i: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 131, nr 23, s. 5795-5805Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The mammalian focal adhesion kinase (FAK) family of non-receptor protein-tyrosine kinases has been implicated in controlling a multitude of cellular responses to the engagement of cell-surface integrins and G-protein-coupled receptors. The high level of sequence conservation between the mammalian proteins and the Drosophila homologue of FAK, Fak56, suggested that it would have similar functions. However, we show here that Drosophila Fak56 is not essential for integrin functions in adhesion, migration or signaling in vivo. Furthermore, animals lacking Fak56 are viable and fertile, demonstrating that Fak56 is not essential for other developmental or physiological functions. Despite this, overexpressed Fak56 is a potent inhibitor of integrins binding to the extracellular matrix, suggesting that Fak56 may play a subtle role in the negative regulation of integrin adhesion.

Nyckelord
Animals, Blotting; Southern, Blotting; Western, Cell Membrane/metabolism, Cytoskeleton/metabolism, DNA/metabolism, Down-Regulation, Drosophila, Drosophila Proteins, Drosophila melanogaster, Focal Adhesion Kinase 1, Focal Adhesion Protein-Tyrosine Kinases, Gene Deletion, Gene Expression Regulation, Immunoblotting, Integrins/*metabolism, Models; Genetic, Muscles/embryology, Mutation, Phenotype, Phosphorylation, Protein Structure; Tertiary, Protein-Tyrosine Kinases/*genetics/*physiology, Signal Transduction, Transgenes, Tyrosine/metabolism
Identifikatorer
urn:nbn:se:umu:diva-17928 (URN)10.1242/dev.01462 (DOI)15525665 (PubMedID)2-s2.0-12344250987 (Scopus ID)
Tillgänglig från: 2007-11-23 Skapad: 2007-11-23 Senast uppdaterad: 2023-03-23Bibliografiskt granskad
2. Mutation of Drosophila focal adhesion kinase induces bang sensitive behaviour and disrupts axonal conduction and synaptic transmission
Öppna denna publikation i ny flik eller fönster >>Mutation of Drosophila focal adhesion kinase induces bang sensitive behaviour and disrupts axonal conduction and synaptic transmission
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Manuskript (Övrigt vetenskapligt)
Identifikatorer
urn:nbn:se:umu:diva-5656 (URN)
Tillgänglig från: 2007-01-19 Skapad: 2007-01-19 Senast uppdaterad: 2010-01-13Bibliografiskt granskad
3. Identification and characterization of DAlk: a novel Drosophila melanogaster RTK which drives ERK activation in vivo.
Öppna denna publikation i ny flik eller fönster >>Identification and characterization of DAlk: a novel Drosophila melanogaster RTK which drives ERK activation in vivo.
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2001 (Engelska)Ingår i: Genes to Cells, ISSN 1356-9597, E-ISSN 1365-2443, Vol. 6, nr 6, s. 531-544Artikel i tidskrift (Refereegranskat) Published
Identifikatorer
urn:nbn:se:umu:diva-4344 (URN)10.1046/j.1365-2443.2001.00440.x (DOI)2-s2.0-0034933673 (Scopus ID)
Tillgänglig från: 2004-11-01 Skapad: 2004-11-01 Senast uppdaterad: 2023-03-24Bibliografiskt granskad
4. Jeb signals through the Alk receptor tyrosine kinase to drive visceral muscle fusion
Öppna denna publikation i ny flik eller fönster >>Jeb signals through the Alk receptor tyrosine kinase to drive visceral muscle fusion
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2003 (Engelska)Ingår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 425, nr 6957, s. 512-516Artikel i tidskrift (Refereegranskat) Published
Identifikatorer
urn:nbn:se:umu:diva-3564 (URN)14523447 (PubMedID)2-s2.0-0141929353 (Scopus ID)
Tillgänglig från: 2008-10-29 Skapad: 2008-10-29 Senast uppdaterad: 2023-03-24Bibliografiskt granskad
5. The Drosophila homologue of the endocytic regulator CIN85 interacts with the RhoGAP RICH1
Öppna denna publikation i ny flik eller fönster >>The Drosophila homologue of the endocytic regulator CIN85 interacts with the RhoGAP RICH1
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Manuskript (Övrigt vetenskapligt)
Identifikatorer
urn:nbn:se:umu:diva-5659 (URN)
Tillgänglig från: 2007-01-19 Skapad: 2007-01-19 Senast uppdaterad: 2010-01-13Bibliografiskt granskad

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