THE REGULATION OF PROTEIN PHOSPHORYLATION

SHYH-LONG CHEN, Purdue University

Abstract

FSBA was used to modify the P-enolpyruvate binding site of pyruvate kinase from rat liver. The Fru-1,6-P(,2) binding site seemed to be modified by FSBA in the presence of P-enolpyruvate. This ATP analog was also used to modify the nucleotide binding sites of acetyl-CoA carboxylase. The studies suggested that carboxylase has three distinct nucleotide binding sites: one cAMP binding site, one catalytic ATP binding site, and one ATP regulatory site. BSA was not generally believed to be phosphorylated by protein kinases. However, BSA was found extensively phosphorylated during the study of the inactivation and phosphorylation of acetyl-CoA carboxylase from rat epididymal fat tissue. The conditions for BSA phosphorylation by the catalytic subunit of the cAMP-dependent protein kinase and the characteristics of phosphate incorporated into BSA were also studied. The BSA phosphorylation required dithiothreitol of GSH. GSSG counteracted the effect of thiols. Phosphorylation increased in a time- and dithiothreitol concentration-dependent manner when BSA was preincubated with dithiothreitol. The incorporation of ('32)P correlated with the appearance of up to six free sulfhydryl groups. Reoxidation of reduced BSA decreased phosphorylation. These experiments showed that this in vitro phosphorylation is dependent on the thiol-disulfide state of BSA. In addition, dithiothreitol and GSH stimulated the phosphorylation of P-93 from both rat liver and epididymal fat tissue and decreased the phosphorylation of P-44 from fat tissue. GSSG decreased the phosphorylation of both P-93 and P-44. The effect of thiols on the protein phosphorylation could be mimicked by NADPH. The increase in sulfhydryl groups of P-93, in response to GSH treatment, was accompanied by an increase in its phosphorylation. These experiments suggested that the reduction of protein disulfides affects the phosphorylation. Some evidence suggested that P-93 is a subunit of phosphorylase. It was also shown that in some other protein phosphorylation systems, the structure change of kinase substrate by dithiothreitol can determine its phosphorylation state. This thesis proposes that a change in thiol-disulfide state of a protein substrate may function as a regulatory mechanism for protein phosphorylation in vitro.

Degree

Ph.D.

Subject Area

Biochemistry

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