Degree Type

Dissertation

Date of Award

1999

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Donald J. Graves

Abstract

An essential mechanism of regulation for many cellular processes is reversible phosphorylation. The phosphorylation state of a protein is determined via a dynamic equilibrium between protein kinase(s) and protein phosphatase(s). A classical example is the interconversion of glycogen phosphorylase. Phosphorylase kinase catalyzes the phosphorylation of glycogen phosphorylase converting it from the inactive b&barbelow; form, to the active a&barbelow; form. Protein phosphatase-1 dephosphorylates phosphorylase a&barbelow; converting it to phosphorylase b&barbelow;. One goal is to understand the specificity of phosphorylase kinase and protein phosphatase-1 for phosphorylase. A second goal is to understand the regulation of phosphorylase kinase by specific inhibitory domains;Previous peptide studies have shown that basic residues N-terminal to the phosphorylatable serine in phosphorylase are known positive determinants for phosphorylase kinase recognition. New studies with peptide substrates indicate that a basic residue C-terminal to the phosphorylatable serine of phosphorylase is another specificity determinant for phosphorylase kinase;Phosphorylase kinase is a member of the calcium/calmodulin regulated protein kinase family. Protein kinases in this family are intrasterically regulated by an autoinhibitory domain in the C-terminus. The C-terminus of phosphorylase kinase, PhK86, has two autoinhibitory regions, PhK13 and PhK5. Residues of PhK13 important for the inhibition of phosphorylase kinase have been identified. All the residues necessary for inhibition are localized in the N-terminal half of PhK13. One residue, cysteine 308, is particularly important. An expression system and purification scheme was developed in an attempt to obtain an intact C-terminus of the catalytic subunit of phosphorylase kinase that could be suitable for future structural studies;Little is known about the substrate specificity of protein phosphatase-1. Phosphorylase a&barbelow; is the best known protein substrate for protein phosphatase-1. Mutants of phosphorylase a&barbelow; were studied with protein phosphatase-1. Recognition of the phosphorylase a&barbelow; mutant, I13G, by protein phosphatase-1 was altered. No detectable activity was observed in this mutant after phosphorylation. The basic residue C-terminal to the phosphoserine residue 14 of phosphorylase a&barbelow; was found to be a positive determinant for protein phosphatase-1 recognition of phosphorylase.

DOI

https://doi.org/10.31274/rtd-180813-13817

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Cheryl Jean Suitt Bartleson

Language

en

Proquest ID

AAI9924700

File Format

application/pdf

File Size

155 pages

Included in

Biochemistry Commons

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