Anti-immunoglobulin M activates nuclear calcium/calmodulin-dependent protein kinase II in human B lymphocytes.
We and others have previously shown that the nuclear protein, Ets-1, is phosphorylated in a calcium-dependent manner after ligation of immunoglobulin (Ig) M on B lymphocytes. As this phosphorylation was independent of protein kinase C activity, we tested whether a calcium/calmodulin-dependent protein kinase (CaM kinase) might phosphorylate the Ets-1 protein after elevation of intracellular free calcium concentrations. The dephosphorylated form of Ets-1 has been shown to bind to chromatin, suggesting that the operative kinase should be detectable in the nucleus. We prepared nuclear extracts from two human B cell lines in which increased intracellular free calcium levels correlated with increased phosphorylation of the Ets-1 protein. Activity of the CaM kinases was determined using a synthetic peptide substrate both in the absence and presence of an inhibitor specific for the CaM kinase family, KN-62. Stimulation of cells with anti-IgM led to increased activity of a nuclear kinase that could phosphorylate the peptide, and this activity was reduced by 10 microM KN-62. Kinase activity was reduced in lysates preadsorbed using an antibody specific for CaM kinase II. Two-dimensional phosphopeptide maps of the Ets-1 protein from cells incubated with ionomycin or anti-IgM contained two unique phosphopeptides that were absent in untreated cells. Incubation of isolated Ets-1 protein with purified CaM kinase II produced phosphorylation of peptides that migrated identically to those found in cells incubated with either anti-IgM or ionomycin. These data suggest a model of signal transduction by the antigen receptor on B lymphocytes in which increased intracellular free calcium can rapidly activate nuclear CaM kinase II, potentially resulting in phosphorylation and regulation of DNA-binding proteins.
D000595:Amino Acid Sequence; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D001402:B-Lymphocytes / Q000201:enzymology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002467:Cell Nucleus / Q000201:enzymology; D002478:Cells, Cultured; D004789:Enzyme Activation; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology; D015759:Ionomycin / Q000276:immunology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D011940:Receptor Aggregation; D011947:Receptors, Antigen, B-Cell / Q000502:physiology*; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*
D000888:Antibodies, Anti-Idiotypic; C494041:ETS1 protein, human; D007075:Immunoglobulin M; D010455:Peptides; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011947:Receptors, Antigen, B-Cell; D014157:Transcription Factors; D015759:Ionomycin; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases