This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phosphorylase kinase (PhK) is a key regulatory enzyme involved in the glycogenolytic pathway, catalyzing the activation, by phosphorylation, of phosphorylase. The hexadecameric enzyme, with subunit composition (alpha,beta,gamma,delta)4, serves to integrate hormonal, metabolic and neural signals, leading to the breakdown of glycogen stores in muscle and liver. Each subunit of PhK is encoded by a separate gene, and multiple tissue specific isoforms exist for all of the subunits. While much work has been done to elucidate the biochemical regulation of PhK catalysis, little information regarding transcriptional regulation of this complex oligomer is available. Our work seeks to elucidate those elements responsible for the temporal and spatial regulation of PhK gene expression by providing for the identification and functional analysis of all human alpha, beta and gamma promoters. The proposed project has four aims directed at this goal. 1) The putative PhK promoter reg ions for all alpha, beta and gamma isoforms will be identified through bioinformatic analysis of the human genome database and amplified by PCR. 2) The minimal sequence necessary to convey spatial and temporal gene expression will be determined for each promoter. 3) Key regulatory cis- and trans-acting factors will be identified for each promoter. 4) Microarray analysis will be performed on RNA from PhK deficient mice to identify novel proteins involved in coordinated gene expression. Knowledge gained from these aims will help determine how PhK expression is orchestrated to ensure that metabolic plasticity is modulated in response to changing energy demands.
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