Homeodomain-interacting Protein Kinase (Hipk) Plays Roles in Nervous System and Muscle Structure and Function

Homeodomain-interacting protein kinases (Hipks) have been previously associated with cell proliferation and cancer, however, their effects in the nervous system are less well understood. We have used Drosophila melanogaster to evaluate the effects of altered Hipk expression on the nervous system and muscle. Using genetic manipulation of Hipk expression we demonstrate that knockdown and over-expression of Hipk produces early adult lethality, possibly due to the effects on the nervous system and muscle involvement. We find that optimal levels of Hipk are critical for the function of dopaminergic neurons and glial cells in the nervous system, as well as muscle. Furthermore, manipulation of Hipk affects the structure of the larval neuromuscular junction (NMJ) by promoting its growth. Hipk regulates the phosphorylation of the synapse-associated cytoskeletal protein Hu-li tai shao (Hts; adducin in mammals) and modulates the expression of two important protein kinases, Calcium-calmodulin protein kinase II (CaMKII) and Partitioning-defective 1 (PAR-1), all of which may alter neuromuscular structure/function and influence lethality. Hipk also modifies the levels of an important nuclear protein, TBPH, the fly orthologue of TAR DNA-binding protein 43 (TDP-43), which may have relevance for understanding motor neuron diseases

Analyse von Wechselwirkungen der Homeodomänen Interagierenden Proteinkinase (Hipk) mit Genen der Augenentwicklung von Drosophila melanogaster

Ziel der Arbeit war die Untersuchung des einzigen bei Drosophila vorkommenden Vertreters der HIPK-Familie im Kontext der Augenentwicklung. Die retinale Determination wird bei Drosophila von den Faktoren des Retinalen Determinations Gen Netzwerkes (RDGN) gesteuert. Während einer Interaktionsstudie mit Hilfe der Bimolekularen Fluoreszenzkomplementation (BiFC) konnten die RDGN-Faktoren Twin of Eyeless (Toy) und Eyeless (Ey) als in vivo Interaktionspartner der Hipk bestätigt werden. Außerdem konnten beide Proteine durch in vitro Phosphorylierungsanalysen als Substrat der Hipk identifiziert werden und die vollständige Kartierung von Toy ergab vier Hipk-Phosphorylierungsstellen. Um die Auswirkungen der Hipk-Phosphorylierung von Toy in vivo zu untersuchen, wurden ausgehend von den kartierten Phosphorylierungsstellen transgene phosphorylierungsmutante Fliegenstämme erzeugt und diese zur ektopischen Expression im Rahmen von Fehlexpressionsanalysen eingesetzt. Zur Auffindung weiterer Wechselwirkungen der Hipk mit Faktoren der Augenentwicklung wurden Kreuzungsanalysen bei variierender Hipk-Dosis durchgeführt. Außerdem konnten bei einer Untersuchung regulatorischer Regionen von hipk drei potentiell augenspezifische hipk-Enhancer identifiziert werden. Zur funktionellen Analyse wurde ein entsprechender transgener Fliegenstamm erzeugt (hipkEGT).HIPKs are well conserved in different signaling pathways and developmental processes throughout the animal kingdom. Purpose of this work was the investigation of the only representative of the Hipk family in Drosophila within the context of eye development. Retinal determination in Drosophila is molecularly controlled by the Retinal Determination Gene Network (RDGN). Using a Bimolecular Fluorescence Complementation (BiFC), the two RDGN factors and Paired box protein 6 (PAX6) homologues Twin of eyeless (Toy) and Eyeless (Ey) were confirmed to be in vivo interaction partners of Hipk. In addition, Toy and Ey were set in an enzyme-substrate relationship with Hipk by in vitro phosphorylation assays. Full mapping of Toy revealed four Hipk phosphorylation sites. In order to investigate the effects of Hipk phosphorylation in vivo transgenic phosphorylation mutant fly strains were generated based on the mapped Hipk-phosphorylation sites and used for ectopic expression. In order to detect further interactions of the Hipk with factors of eye development, genetic analyzes were performed at varying Hipk dose. In addition, an analysis of hipk regulatory regions by larval reporter gene expression identified three specific hipk enhancers for expression in developing eye tissue. For the functional analysis a corresponding transgenic fly strain was generated (hipkEGT)

Identification of Direct Substrates for CMGC Kinases with 18O-ATP-based Kinase Assay and LC-MS/MS

Protein kinases are signaling molecules that regulate vital cellular and biological processes by phosphorylating cellular proteins. Kinases are linked to variety of diseases such as cancer, immune deficiencies and degenerative diseases. This thesis work aimed to identify direct substrates for protein kinases in the CMGC family, which consists of the cyclin-depended kinases (CDK), mitogen activated protein kinases (MAPK), glycogen synthase kinase-3 (GSK3) and CDC-like kinases (CLK). CMGC kinases have been identified as cancer hubs in interactome studies, but large-scale identification of direct substrates has been difficult due to the lack of efficient methods. Here, we present a heavy-labeled 18O-ATP-based kinase assay combined with LC-MS/MS analysis for direct substrate identification. In the assay, HEK and HeLa cell lysates are treated with a pan-kinase inhibitor FSBA which irreversibly blocks endogenous kinases. After the removal of FSBA, cell lysates are incubated with the kinase of interest and a heavy-labeled ATP, which contains 18O isotope at the γ-phosphate position. Resulting phosphopeptides are enriched with Ti4+- IMAC before the LC-MS/MS analysis, which distinguishes the desired phosphorylation events based on a mass shift caused by the heavy 18O. With this pipeline of methods, we managed to quantify and identify direct substrates for 26 members of CMGC kinase family. A total of 1345 substrates and 3841 interacting kinase-substrate pairs were identified in cytosolic cell lysates, from which 165 were annotated in the PhosphoSitePlus® database. To identify substrates for kinases with nuclear localization, ten kinases were tested with nuclear HEK cell lysate. We identified 194 kinase-substrate pairs, 141 of which were unique to the nuclear fraction and 27 annotated in the PhosphoSitePlus® database. Finally, kinases with outstandingly high amounts of novel substrates were subjected to gene ontology analysis. We were able to link the gene ontology classifications of novel substrates to the biological processes regulated by the kinase of interest. These results indicate that heavy-labeled 18O-ATP-based kinase assay linked LC-MS/MS is a useful tool for large-scale direct kinase substrate identification

Genetic Modifier Screens Reveal New Components that Interact with the Drosophila Dystroglycan-Dystrophin Complex

The Dystroglycan-Dystrophin (Dg-Dys) complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, we used a model organism Drosophila melanogaster and performed genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-β and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought