Differential phosphorylations of Spi-B and Spi-1 transcription factors.

Spi-1/PU-1 and Spi-B are hematopoietic transcription factors, which, in vitro, display similar affinities for DNA target sequences containing the consensus binding site 5'-GGAA-3'. While the role of Spi-1 in the transcriptional regulation of B cell and myeloid specific genes has been largely demonstrated, the biological function of Spi-B still remains ...
to be elucidated. Since Spi-B and Spi-1 are very divergent in their transactivator domain, these domains might acquire functional specificity in vivo by interacting with different co-factors and/or by undergoing different phosphorylations. First, we observed that casein kinase II phosphorylates Spi-B as well as Spi-1, in vitro. Then, by affinity chromatographies and in vitro kinase assays with fusion proteins between glutathione-S-transferase and the transactivator domain of Spi-B, two kinases were identified on their ability to interact and phosphorylate this domain; the MAP kinase ERK1 and the stress activated protein kinase JNK1. The Threonine 56 was defined as the ERK1 phosphorylation site by using phosphoamino-acid analyses and a Spi-B mutant version with the substitution T56 to A56. Strikingly, ERK1 failed to phosphorylate Spi-1, in vitro, whereas JNK1, like CK II, phosphorylated Spi-B and Spi-1. In addition, other purified Spi-B-kinase activities, unidentified as yet, display similar specificity than ERK1 for Spi-B versus Spi-1. Furthermore, the evident interaction of pRb protein with the transactivator domain of Spi-B in an unphosphorylated state disappeared when this domain was first phosphorylated in vitro either by ERK1 or by the purified Spi-B-kinase activities. Our data revealed multiple phosphorylation sites within Spi-B whose some of them appeared specific for Spi-B versus Spi-1 and which may account for differential regulation of their activities.
Mesh Terms:
Amino Acid Sequence, Animals, Blotting, Western, Burkitt Lymphoma, Calcium-Calmodulin-Dependent Protein Kinases, Cell Line, Cell Nucleus, Cercopithecus aethiops, Chromatography, Affinity, DNA-Binding Proteins, Genes, Retinoblastoma, Glutathione Transferase, Humans, JNK Mitogen-Activated Protein Kinases, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinases, Molecular Sequence Data, Phosphorylation, Protein Biosynthesis, Recombinant Fusion Proteins, Retinoblastoma Protein, Retroviridae Proteins, Oncogenic, Substrate Specificity, Transcription Factors, Transcription, Genetic, Transfection, Tumor Cells, Cultured
Oncogene
Date: Feb. 15, 1996
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