calpain and Leukemia--Myeloid

Human recombinant CK2 subunits were incubated for different times with the two main cytosolic proteases m-calpain and 20 S proteasome. Both, m-calpain in a calcium dependent manner and the 20 S proteasome, were able to degrade CK2 subunits in vitro. In both cases, CK2alpha' was more resistant to these proteases than CK2alpha. When these proteases were assayed on the reconstituted (alpha2beta2 holoenzyme), a 37 kDa alpha-band, analogous to that observed in AML extracts, was generated which was resistant to further degradation. No degradation was observed when the 26 S proteasome was assayed on free subunits. Studies with CK2alpha deletion mutants showed that m-calpain and the 20 S proteasome acted on the C-terminus end of CK2alpha. These results pointed to cytosolic proteases as agents involved in the control of the amount of free CK2 subunits within the cell, which becomes evident when CK2 is overexpressed as in AML cells.

Topics: Acute Disease; Amino Acid Sequence; Animals; Calpain; Casein Kinase II; Cysteine Endopeptidases; HL-60 Cells; Humans; Hydrolysis; Isoenzymes; Leukemia, Myeloid; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Rabbits; Recombinant Proteins

Cleavage of specific protein subsets is a key event in the execution of apoptosis. Protein degradation may serve for the structural alterations that result in cell self-destruction, but it may also function as a switch in the decisions between apoptosis and necrosis or apoptosis and cell proliferation. Here, we show that MCM3, but not other members of the Mcm family of replicative proteins, is cleaved early in several models of apoptosis. Cleavage of MCM3 can be prevented by caspase inhibitors, and it does not occur when cells are forced to undergo necrosis by energy deprivation. We propose that active destruction of MCM3 inactivates the Mcm complex and serves to prevent untimely DNA replication events during the execution of the cell death program.

Topics: Apoptosis; Calpain; Cell Cycle Proteins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Fragmentation; DNA-Binding Proteins; fas Receptor; Humans; Jurkat Cells; Leukemia, Myeloid; Minichromosome Maintenance Complex Component 3; Nuclear Proteins; Serine Proteinase Inhibitors; Tumor Cells, Cultured; Zinc Sulfate

To clarify phosphorylation of calpains I and II in vivo, we purified both calpains concurrently from the [32P] metabolic-labeled human chronic myelogenous leukemia cell line K-562. By Ultragel AcA34 column chromatography, enzymatic activity of calpain I was separated from [32P] radioactivity. Whereas calpain II activity was closely associated with [32P] radioactivity on Ultragel AcA34 and Blue Sepharose CL-6B column chromatographies. By the above purification procedures, calpain I was purified 1300-fold from the crude extract and calpain II was 920-fold from the original sample, respectively. Autoradiographies of purified calpains I and II from [32P] labeled K-562 cells revealed that both calpains were not specifically phosphorylated in vivo. The autophosphorylation in vitro on calpains and modulation of their proteolytic activities reported recently thus may not occur within cells.

Topics: Autoradiography; Calpain; Cell Line; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Humans; Leukemia, Myeloid; Molecular Weight; Phosphorylation