Cloning & Cellular Characterization of Myosin II Heavy Chain Kinase D from Dictyostelium discoideum.

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Travis R. Russell (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Advisor
Paul Steimle

Abstract: The thesis research presented here focused on studies of a novel myosin II heavy chain kinase D (MHCK D) expressed in Dictyostelium discoideum cells. MHCK D is made up of four distinct domains: a short coiled-coil region (Coil), a region rich in serine, asparagine, proline, & glutamine residues (SNPQ), a kinase catalytic domain (Cat), and a WD-repeat segment (WD). A major component of this project was to amplify the Catalytic and WD repeat domains (Cat-WD) from genomic DNA. The Cat-WD truncation of MHCK D was cloned into pTX-GFP & pTX-Flag plasmids for expression in Dictyostelium cells. The recombinant plasmids were electroporated into Dictyostelium cells to over-express the fusion protein (GFP, or flag-tagged). To determine if MHCK D does phosphorylate the Myosin heavy chain, which in turn drives bi-polar filament disassembly, the phenotype of cells overexpressing the Cat-WD domain were compared to the phenotype of Myosin II-null cells and non-recombinant wild type cells. Cells overexpressing the Cat-WD domain from MHCK D, showed a phenotype similar to Myosin II-null cells, indicating that the Cat-WD domain plays a role in Myosin II bi-polar filament disassembly resulting from phosphorylation of the myosin heavy chain. Another aim of the thesis research focused on expression of the mhkd gene during the multicellular development cycle of Dictyostelium, using Reverse Transcriptase-PCR (RTPCR). The results indicate that mhkd is expressed throughout the multi-cellular development cycle, as well as during the vegetative growth phase of Dictyostelium. In summary, the results reveal that the Cat-WD domain of MHCK D drives Myosin II bipolar filament disassembly leading to defects in cytokinesis, and is expressed in both vegetative cells as well as cells undergoing the multi-cellular development cycle. These studies provide a basis for future research focused on activation, localization, and substrate targeting of MHCK D.

Additional Information

Publication
Thesis
Language: English
Date: 2008
Keywords
Dictyostelium, Myosin, Kinase, Protein, MHCK D, MHC
Subjects
Dictyostelium discoideum $x Physiology.
Cytokinesis $x Physiology.
Cytoskeletal proteins.
Myosin.
Protein kinases.