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.
Mao
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Unité INSERM 248, Faculté de Médecine Lariboisière-Saint Louis, Paris, France.
Ray-Gallet
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Tavitian
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Moreau-Gachelin
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D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002051:Burkitt Lymphoma; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016161:Genes, Retinoblastoma; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000096:biosynthesis; D015682:Retroviridae Proteins, Oncogenic; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D015682:Retroviridae Proteins, Oncogenic; D014157:Transcription Factors; C062022:v-Spi-1 protein, Friend spleen focus-forming virus; C076877:SPIB protein, human; D005982:Glutathione Transferase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases