Protein kinase A and C site-specific phosphorylations of LAP (NF-IL6) modulate its binding affinity to DNA recognition elements.

LAP (NF-IL6 or C/EBP beta), is a liver transcriptional activator protein that confers liver-specific gene expression. Because LAP has a characteristic phosphoacceptor sequence for cAMP-dependent protein kinase A (PKA), we tested if in vitro phosphorylation of LAP by PKA modulates its interaction with specific DNA sequences. The major PKA phosphorylation site of LAP was identified as Ser105, which is a predicted PKA site. As expected, this PKA phosphorylation site disappears after mutation of Ser105 to Ala. Kinetic studies with LAP and LAP Asp105 (which mimics a phosphoserine residue) demonstrated that phosphorylation of Ser105 itself has no effect on DNA binding. Phosphorylation of other sites by PKA, identified in the region between Ser173 and Ser223 and at Ser240, by analysis of truncated and mutated LAP peptides, resulted in an inhibition of DNA binding. LAP was also phosphorylated by purified protein kinase C in vitro, and the major phosphoacceptor was shown to be Ser240 within the DNA-binding domain of LAP. Phosphorylation of LAP at this residue or introduction of a Ser240 to Asp mutation resulted in marked decrease in its binding to DNA. These results suggest that site-specific phosphorylations of LAP modulate transactivation of its target genes.

[1]  J. Järv,et al.  Substrate specificity of protein kinase C studied with peptides containing D-amino acid residues. , 1993, Journal of biochemistry.

[2]  M. Rosenfeld,et al.  Calcium-Regulated Phosphorylation Within the Leucine Zipper of C/EBPβ , 1992, Science.

[3]  M. Karin,et al.  Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73 , 1991, Nature.

[4]  James R. Woodgett,et al.  Phosphorylation of c-jun mediated by MAP kinases , 1991, Nature.

[5]  E. Ziff,et al.  cAMP stimulates the C/EBP-related transcription factor rNFIL-6 to trans-locate to the nucleus and induce c-fos transcription. , 1991, Genes & development.

[6]  R. Umek,et al.  Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. , 1991, Genes & development.

[7]  R. Weinberg,et al.  G1/S phosphorylation of the retinoblastoma protein is associated with an altered affinity for the nuclear compartment , 1991, Cell.

[8]  Tony Hunter,et al.  Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity , 1991, Cell.

[9]  T. Hunter,et al.  Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. , 1991, Methods in enzymology.

[10]  R. Cortese,et al.  IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C EBP , 1990, Cell.

[11]  U. Schibler,et al.  LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein. , 1990, Genes & development.

[12]  S. Akira,et al.  A nuclear factor for IL‐6 expression (NF‐IL6) is a member of a C/EBP family. , 1990, The EMBO journal.

[13]  M. Montminy,et al.  Characterization of a bipartite activator domain in transcription factor CREB , 1990, Cell.

[14]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[15]  M. Montminy,et al.  Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133 , 1989, Cell.

[16]  M. Montminy,et al.  Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB , 1988, Nature.

[17]  S. McKnight,et al.  Isolation of a recombinant copy of the gene encoding C/EBP. , 1988, Genes & development.

[18]  Ueli Schibler,et al.  The interplay of DNA-binding proteins on the promoter of the mouse albumin gene , 1987, Cell.

[19]  A. Panduro,et al.  Changing patterns of transcriptional and post-transcriptional control of liver-specific gene expression during rat development. , 1987, Genes & development.

[20]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.