Studies on the phosphorylation of a signal peptide involved in apoptosis induced by antibody in the human leukemia cell line, K-562.

Author

James W. Hodge

Date of Award

5-1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Carl J. Wust

Committee Members

A. Ichiki, W. Farkas, C. Lozzio

Abstract

Gamma globulin from rabbit anti-K-562 serum and certain monoclonal antibodies reactive with the human leukemia cell line, K-562, induce a sequence of events beginning with decreased DNA, RNA and protein synthesis within minutes of antibody addition and leading to eventual cell death without lysis in 2 to 4 days (Leukemia Research 15, 497, 1991). The overall sequence is analogous to that observed in a form of programmed cell death, referred to as apoptosis, including the cardinal signs of chromatin condensation, formation of apoptotic bodies and single strand DNA nicking.

We examined possible signal transductions that might be initiated by antibody. After addition of the antibody, there is an elevation of ATP (3 fold over controls) within 15 minutes. The ATP concentration in antibody-treated cells falls rapidly to below detectable levels by 90 minutes and precedes the appearance of a phosphorylated molecule of less than 2000 Daltons. The amount of the latter product peaks at 60 minutes and decreases to less than 5% of the peak value by 240 minutes. This phosphorylated product in turn precedes all other cardinal signs of apoptosis in this system. We have determined that the low molecular weight product is a peptide, and have named it Hemlock Factor due to its putative role in the death of the cell.

Hemlock Factor-peptide is composed of twelve amino acids: 8 tyrosines, 1 serine, 1 glycine, 1 tryptophan, and 1 proline. The peptide is present in equimolar concentrations in K-562 (276 picograms/cell) before and after the cells are induced to die by the addition of antiserum. Hemlock Factor is associated with the cell membrane before treatment and becomes cytosolic after antiserum addition. The peptide becomes hyperphosphorylated on the tyrosine residues after antibody treatment, and this phosphorylation is coincident with the inactivation of the mitochondria. The amino terminus of the peptide is blocked and an exact amino acid sequence is unavailable to date. Because the peptide contains 8 residues of tyrosine, a Genbank™ query yields a strong sequence homology (75%) to the region of the variable domain within residue positions 101 to 112 of the immunoglobulin heavy chain, and to several viral immediate early (IE) proteins (75% homology). The peptide is readily isolated from antibody-treated K-562 cells, and it has been isolated from seven different sublines of K-562, but not from normal human peripheral blood cells or ten other neoplastic cell lines. The isolation procedure yields approximately 350μg peptide/6 x 10⁶ cells with a greater than 90% purity as determined by High Pressure Liquid Chromatography.

Exogenously purified Hemlock Factor from antibody-treated cells is toxic for K-562 cells in a dose dependent manner and acts synergistically with anti-K-562 serum. An effect of the peptide on other cell lines has not been fully established owing to the probable lack of a transmembrane transport system for the peptide.

The results from this report, taken together, support the model that K-562 cells are constitutively synthesizing a small molecular weight peptide, which we call Hemlock Factor. This peptide is present in large amounts in the cell (~9.6 x 10¹⁰ molecules per cell) and is normally bound to the cytoplasmic face of the plasma membrane by the covalent linkage to a fatty acid such as myristic acid. Upon the addition of antiserum, the complexing of antibody to the antigen receptor directly or indirectly activates a deacetylase culminating in the release of the peptide into the cytoplasm. The Hemlock Factor-peptide is then available to become hyperphosphorylated by a resident tyrosine kinase (there are over 20 normally active in K-562 cells). The low molecular weight of the peptide (1732 Da) allows it to be synported into the mitochondria, but due to the high negative charge, more hydrogen ions from the mitochondrial membrane are needed to neutralize the charge than with a normal substrate. The mitochondrial membrane potential becomes destroyed and the ATP synthesizing enzyme, F₀F₁ ATPase, looses the ability to synthesize ATP. The depletion of ATP in the K-562 cell would inhibit certain enzymes, such as DNA and RNA polymerases, and thus DNA and RNA synthesis; aminoacyl-tRNA synthetases, and thus protein synthesis; actin polymerase, and thus cytoskeletal integrity; and DNA repair/ligation enzymes coinciding with the accumulation of DNA damage in the form of free translocation elements characteristic of this cell line.

The data suggests that some surface receptors react with antibody to transduce signals which lead to the phosphorylation of the second messenger, Hemlock Factor peptide, which initiates the cascade resulting in programmed cell death.

Recommended Citation

Hodge, James W., "Studies on the phosphorylation of a signal peptide involved in apoptosis induced by antibody in the human leukemia cell line, K-562.. " PhD diss., University of Tennessee, 1993.
https://trace.tennessee.edu/utk_graddiss/10688