Date of Award

1-21-1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Harvey R. Knull

Abstract

Glycolytic enzymes have been shown to bind to the cytoskeletal protein, tubulin. These protein-protein interactions have been hypothesized to provide compartmentation of glycolysis in the cytoplasm of eukaryotic cells via the cytoskeleton, a component of the microtrabecular lattice.Previously, in my thesis work, it was determined that four enzymes of glycolysis (glyceraldehyde-3-phosphate dehydrogenase, aldolase, pyruvate kinase, and lactate dehydrogenase-muscle type) had high affinity for the $\alpha$-subunit C-terminal region of tubulin. When this region was removed proteolytically, binding was diminished. The localization of the glycolytic enzyme domain on tubulin, phosphorylation of this domain, and in vivo effects of the domain are investigated herein in three separate but congruent studies.There were three objectives in the first study. First, the glycolytic enzyme binding domain from native tubulin was isolated from chemically cleaved tubulin. Second, different length peptides were synthesized to further characterize the binding domain. Third, the effectiveness of the synthetic peptides towards enzyme binding was determined. A peptide was chemically cleaved from native tubulin. It was isolated by DEAE-HPLC, its binding parameters were characterized with aldolase, and its sequence was determined. It was discovered that the peptide was from the C-terminal of the tubulin $\alpha$-subunit. Synthetic peptides were prepared representing different length fragments of this region. A peptide containing the last 43 amino acids had the same binding parameters as native tubulin and was therefore designated the tubulin glycolytic enzyme binding domain (T-GEBD-43mer). No secondary structure was detected by circular dichroism of the T-GEBD-43mer, suggesting that secondary structure may not be important for the enzymes to bind.There were three objectives in the second study. First, the effects of phosphorylation of tubulin by ZAP-70, a T-leukocyte protein-tyrosine kinase (PTK), on binding between tubulin and the glycolytic enzymes were investigated. Second, the effects of phosphorylation of T-GEBD-43mer by ZAP-70 were determined. Third, the effect of T-GEBD-43mer in L-929 cells was determined. Phosphorylation of both tubulin and the T-GEBD-43mer caused reduced interactions between tubulin and T-GEBD-43mer. In vivo introduction of the T-GEBD-43mer caused drastic suppression of glycolysis as measured by reduced lactate production.There were three objectives in the third study. First, it was determined that the epidermal growth factor (EGF) receptor, a PTK, recognized the T-GEBD-43mer as a substrate. Second, kinetic parameters of EGF receptor for phosphorylation of T-GEBD-43mer by the EGF receptor were determined. Third, as in study two, enzyme binding to T-GEBD-43mer after phosphorylation was determined. Enzyme kinetic studies revealed that T-GEBD-43mer was a substrate for EGF receptor. A K$\rm\sb {M}$ of approximately 5.0 $\mu$M suggests that tubulin is a substrate in vivo. As predicted from study two, phosphorylation of T-GEBD-43mer disrupted binding to the enzymes of glycolysis.From the results, it was concluded that the enzymes of glycolysis bind to a 43 amino acid domain located at the C-terminal of the tubulin $\alpha$-subunit. Phosphorylation of this domain may act as a novel regulatory step in glycolysis which has implications in both normal and cancer cells.

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