Effect of PU.1 phosphorylation on interaction with NF-EM5 and transcriptional activation.
PU.1 recruits the binding of a second B cell-restricted nuclear factor, NF-EM5, to a DNA site in the immunoglobulin kappa 3' enhancer. DNA binding by NF-EM5 requires a protein-protein interaction with PU.1 and specific DNA contacts. Dephosphorylated PU.1 bound to DNA but did not interact with NF-EM5. Analysis of serine-to-alanine mutations in PU.1 indicated that serine 148 (Ser148) is required for protein-protein interaction. PU.1 produced in bacteria did not interact with NF-EM5. Phosphorylation of bacterially produced PU.1 by purified casein kinase II modified it to a form that interacted with NF-EM5 and that recruited NF-EM5 to bind to DNA. Phosphopeptide analysis of bacterially produced PU.1 suggested that Ser148 is phosphorylated by casein kinase II. This site is also phosphorylated in vivo. Expression of wild-type PU.1 increased expression of a reporter construct containing the PU.1 and NF-EM5 binding sites nearly sixfold, whereas the Ser148 mutant form only weakly activated transcription. These results demonstrate that phosphorylation of PU.1 at Ser148 is necessary for interaction with NF-EM5 and suggest that this phosphorylation can regulate transcriptional activity.
Pongubala
J M
JM
Department of Animal Biology, University of Pennsylvania, School of Veterinary Medicine, Philadelphia 19104.
Van Beveren
C
C
Nagulapalli
S
S
Klemsz
M J
MJ
McKercher
S R
SR
Maki
R A
RA
Atchison
M L
ML
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010766:Phosphorylation; D010954:Plasmacytoma; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D007145:Immunoglobulin kappa-Chains; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D015682:Retroviridae Proteins, Oncogenic; D014157:Transcription Factors; C062022:v-Spi-1 protein, Friend spleen focus-forming virus