Expression of two 50 kDa proteins is associated with sensitivity towards etherlipid analogues in the human leukaemia cell line HL 60.

Hexadecylphosphocholine (HePC) is a new etherphospholipid derived substance with pronounced antineoplastic activity. So far the mode of action of this compound has not been resolved. Therefore, we decided to approach this problem by generating HePC resistant sublines of susceptible cells. The human leukaemia cell line HL 60 was successfully adapted to high concentrations of HePC over a period of 14 months. The resistant cell line HL 60 R shows similar functional characteristics as the original HL 60. Both lines can be induced to terminal differentiation into a granulocytic phenotype by DMSO. In this process, normal HL 60 cells also become resistant towards HePC. Determinations of cellular membrane lipid composition did not show significant changes, which would explain the resistance mechanism. Analysis of cellular proteins by 2D-gelelectrophoresis revealed two 50 kDa proteins expressed in HL 60 and differentiated HL 60 cells, which were not expressed in HL 60 R. Reversion of resistance of HL 60 R after prolonged cultivation without HePC led to re-expression of the two proteins, indicating at a possible involvement of these proteins in HePC sensitivity.

Are calcium-dependent protein kinases involved in the regulation of glycolytic/gluconeogenetic enzymes? Studies with Ca2+/calmodulin-dependent protein kinase and protein kinase C.

Changes in glycolytic flux have been observed in liver under conditions where effects of cAMP seem unlikely. We have, therefore, studied the phosphorylation of four enzymes involved in the regulation of glycolysis and gluconeogenesis (6-phosphofructo-1-kinase from rat liver and rabbit muscle; pyruvate kinase, 6-phosphofructo-2-kinase and fructose-1,6-bisphosphatase from rat liver) by defined concentrations of two cAMP-independent protein kinases: Ca2+/calmodulin-dependent protein kinase and Ca2+/phospholipid-dependent protein kinase (protein kinase C). The results were compared with those obtained with the catalytic subunit of cAMP-dependent protein kinase. The following results were obtained. 1. Ca2+/calmodulin-dependent protein kinase phosphorylates 6-phosphofructo-1-kinase and L-type pyruvate kinase at a slightly lower rate as compared to cAMP-dependent protein kinase. 2. 6-Phosphofructo-1-kinase is phosphorylated by the two kinases at a single identical position. There is no additive phosphorylation. The final stoichiometry is 2 mol phosphate/mol tetramer. The same holds for L-type pyruvate kinase except that the stoichiometry with either kinase or both kinases together is 4 mol phosphate/mol tetramer. 3. Rabbit muscle 6-phosphofructo-1-kinase is phosphorylated by cAMP-dependent protein kinase but not by Ca2+/calmodulin-dependent protein kinase. 4. Fructose-1,6-bisphosphatase from rat but not from rabbit liver is phosphorylated at the same position but at a markedly lower rate by Ca2+/calmodulin-dependent protein kinase when compared to the phosphorylation by cAMP-dependent protein kinase. 5. 6-Phosphofructo-2-kinase is phosphorylated by Ca2+/calmodulin-dependent protein kinase only at a negligible rate. 6. Protein kinase C does not seem to be involved in the regulation of the enzymes examined: only 6-phosphofructo-2-kinase became phosphorylated to a significant degree. In contrast to the phosphorylation by cAMP-dependent protein kinase, this phosphorylation is not associated with a change of enzyme activity. This agrees with our observation that the sites of phosphorylation by the two kinases are different. The results indicate that Ca2+/calmodulin-dependent protein kinase but not protein kinase C could be involved in the regulation of hepatic glycolytic flux under conditions where changes in the activity of cAMP-dependent protein kinase seem unlikely.