The protein phosphatases are key enzymes in the regulation of protein function by phosphorylation-dephosphorylation mechanisms. The long-standing interests of this laboratory have been in the study of the properties, structure and regulation of the phosphatases involved in the modification of the enzymes of glycogen metabolism, with a specific emphasis on phosphorylase phosphatase. Current evidence indicates the existence of several isoforms of the catalytic subunit of phosphorylase phosphatase- two different cDNAs coding for proteins of 37.5 and 35.4 kDa have been cloned, and sequence studies of the isolated enzyme suggests that there is at least one other isoform. The number of isoforms, their structures and functional properties remain to be rigorously defined. The existence of these isoforms may reflect one or more of the following possibilities: a) they may represent isoforms with different substrate specificities or other functional differences b) they may bind to different regulatory proteins c) they may be differentially expressed in different muscle types, or different tissues, i.e. they are tissue-specific isoforms; d) they may be developmentally regulated; e) they may be under differential hormonal regulation. The genetic basis of the existence of these isoforms is an important questions because the structures of the two known cDNAs suggests the possibility that the isoform mRNAs are derived from a single gene by alterative RNA splicing. Thus, the existence of isoforms of ppasel may be of important functional and physiological significance. This research proposal addresses these questions by the following experimental approaches: 1) separation and characterization of the isoforms with regard to specificity, interaction with regulatory proteins, and subcellular derivation; 2) cDNA cloning to define their primary structures; 3) investigation of the expression of the isoform mRNAs in different muscle types and tissues, and in muscle at different developmental stages; 4) mapping of the intron-exon organization of the gene for the enzyme, and the structural examination of specific regions of the gene by sequencing of genomic clones; 5) expression of the enzyme in a procaryotic vector to develop a means of probing structure-function relationships by mutagenesis. The determination of their primary structures and comparative properties of these isoforms any also provide significant information about their structure-function relationships. The detailed understanding of the properties and regulation of the phosphatases is central to a fuller understanding of how protein phosphorylation mechanisms regulate biological processes. Such information may in the future have a direct impact on our understanding on pathological disease processes, particularly those which involve imbalances in carbohydrate metabolism, including those of diabetes.
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