Athanasios G. Papavassiliou

Intramolecular signal transduction in c-Jun.

The DNA‐binding activity of c‐Jun is determined by the phosphorylation state of a cluster of threonine and serine residues located near its COOH‐terminus. We have analyzed the events that lead to c‐Jun activation via dephosphorylation of these sites in response to phorbol esters. Our results indicate that COOH‐terminal dephosphorylation is an indirect consequence of a separate phosphorylation event targeted to the NH2‐terminus of c‐Jun. Thus, the activation of c‐Jun DNA‐binding potential, caused by COOH‐terminal dephosphorylation, may not require the regulation of the kinase/phosphatase system that brings about this change, but rather an alteration in the accessibility of the COOH‐terminal phosphoacceptor sites of c‐Jun.

Phosphorylation of Drosophila Jun by the MAP kinase rolled regulates photoreceptor differentiation.

Drosophila Jun (D‐Jun) is a nuclear component of the receptor tyrosine kinase/Ras signal transduction pathway which triggers photoreceptor differentiation during eye development. Here we show that D‐Jun is a substrate for the ERK‐related Drosophila MAP kinase Rolled, which has previously been shown to be a part of this pathway. A D‐Jun mutant that carries alanines in place of the Rolled phosphorylation sites acts as a dominant suppressor of photoreceptor cell fate if expressed in the eye imaginal disc. In contrast, a mutant in which the phosphorylation sites are replaced by phosphate‐mimetic Asp residues, as well as a VP16‐D‐Jun fusion protein, can promote photoreceptor differentiation. These data implicate Jun phosphorylation in the choice between neuronal and non‐neuronal fate during Drosophila eye development.

Determining the effect of inducible protein phosphorylation on the DNA-binding activity of transcription factors

Inducible phosphorylation or dephosphorylation of transcription factors is an important mechanism of signal-dependent gene regulation in eukaryotic cells. This paper describes a combination of techniques that can be used to study the effect of this covalent protein modification on the DNA-binding activity of transcription factors. The protein of interest is genetically tagged with oligohistidine to allow rapid purification on nickel-chelate columns after being transiently overexpressed in eukaryotic tissue culture cells. Phosphorylation-dependent DNA-binding activity is determined in a blotting assay including an in situ dephosphorylation step. Studies on the protooncogene-encoded transcription factor c-Jun employing this assay revealed a TPA-inducible protein dephosphorylation event that strongly increases the DNA-binding potential of the protein. Our data confirm the results published recently by others and provide a rapid, efficient, sensitive, and well-controlled experimental system to analyze the phosphorylation-regulated modulations in the DNA-binding activity of transcription factors.