Characterization of dystrophin and utrophin expression and identification of novel associated proteins.

Dystrophin and utrophin are homologous membrane associated cytoskeletal proteins that are required for proper muscle function. These proteins bind to a protein complex at the muscle sarcolemma that links with the extracellular matrix and signalling proteins. Dystrophin and utrophin are also produced in non-muscle tissues; however, less is known about their function in this context. This dissertation focuses on the characterization of dystrophin and utrophin in the mouse. Comparisons between dystrophin and utrophin expression revealed conservation of many features of the dystrophin gene in the utrophin gene. Utrophin was shown to be widely expressed in adult tissues and several putative small utrophin isoforms were identified. Also, experiments determined that the COOH-terminal domain of utrophin was sufficient for associations with neuromuscular junctions. Analysis of the major nonmuscle dystrophin isoform (Dp71) by gene replacement with a reporter gene detected cell specific expression of the Dp71 promoter in the central nervous system, retina, and kidney. In an effort to further define the properties of the dystrophin associated cytoskeleton, the yeast two-hybrid system was utilized to identify novel components of the dystrophin and utrophin complexes. Several new proteins were identified as members of the utrophin associated protein complex via their interactions with beta2-syntrophin, a neuromuscular junction-specific utrophin-associated protein. Two of these proteins, MAST205 (microtubule associated serine/threonine kinase-205 kDa) and SAST (syntrophin associated serine/threonine kinase), were found to be microtubule associated serine/threonine kinases. This study presents the first evidence for an association of a kinase with the dystrophin/utrophin network. MAST205 and SAST localize to postsynaptic regions in muscle and neuronal tissues along with dystrophin and utrophin. Furthermore, evidence was found for associations between syntrophin, dystrophin, and utrophin and microtubules. MAST205 and SAST were found to be members of a larger family of genes called the STP gene family (serine/threonine kinase and PDZ domain). The expression pattern of the STP genes was characterized. The SAST gene was mapped to gene dense regions of mouse chromosome 8 and human chromosome 19p13.2. The identification of novel dystrophin/utrophin associated proteins suggests mechanisms by which the dystrophin network can be regulated and coordinated with other cytoskeletal components.Ph.D.Biological SciencesCellular biologyGeneticsMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132415/2/9963842.pd