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Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts
S J Baker1, T K Kerppola, D Luk
1Department of Molecular Oncology and Virology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110.
Abstract:
c-jun is a member of the family of immediate-early genes whose expression is induced by factors such as serum stimulation, phorbol ester, and differentiation signals. Here we show that increased Jun synthesis after serum stimulation is accompanied by a concomitant increase in phosphorylation. Several serine-threonine kinases were evaluated for their ability to phosphorylate Jun in vitro. p34cdc2, protein kinase C, casein kinase II, and pp44mapk phosphorylated Jun efficiently, whereas cyclic AMP-dependent protein kinase and glycogen synthase kinase III did not. The sites phosphorylated by p34cdc2 were similar to those phosphorylated in vivo after serum induction. The major sites of phosphorylation were mapped to serines 63, 73, and 246. Phosphorylation of full-length Jun with several kinases did not affect the DNA-binding activity of Jun homodimers or Fos-Jun heterodimers. Comparison of the DNA binding and in vitro transcription properties of wild-type and mutated proteins containing either alanine or aspartic acid residues in place of Ser-63, -73, and -246 revealed only minor differences among homodimeric complexes and no differences among Fos-Jun heterodimers. Thus, phosphorylation of Jun did not produce a significant change in dimerization, DNA-binding, or in vitro transcription activity. The regulatory role of phosphorylation in the modulation of Jun function is likely to be considerably more complex than previously suggested.
Insights
Jun protein phosphorylation, induced by serum, does not significantly alter its DNA-binding or transcription activity. This suggests a more complex regulatory role for Jun phosphorylation in gene expression.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Gene Regulation
Background:
- c-Jun is an immediate-early gene transcription factor.
- Jun expression increases with serum stimulation and differentiation signals.
- Post-translational modification, like phosphorylation, can regulate transcription factor activity.
Purpose of the Study:
- To investigate the effect of Jun phosphorylation on its DNA-binding and transcription activity.
- To identify kinases capable of phosphorylating Jun in vitro.
- To map the specific phosphorylation sites on the Jun protein.
Main Methods:
- In vitro kinase assays using various serine-threonine kinases (p34cdc2, PKC, CKII, pp44mapk, PKA, GSK3).
- Phosphorylation site mapping using p34cdc2 and in vivo phosphorylated Jun.
- Electrophoretic mobility shift assays (EMSAs) to assess DNA-binding activity of wild-type and mutant Jun.
- In vitro transcription assays with wild-type and mutated Jun proteins.
Main Results:
- Several kinases (p34cdc2, PKC, CKII, pp44mapk) efficiently phosphorylated Jun in vitro.
- Major phosphorylation sites were identified at Serines 63, 73, and 246.
- Phosphorylation did not significantly alter DNA-binding or in vitro transcription activity of Jun homodimers or Fos-Jun heterodimers.
Conclusions:
- Jun phosphorylation by specific kinases does not directly enhance its DNA-binding or transcription-activating functions.
- The regulatory role of Jun phosphorylation in modulating its function is complex and not fully explained by direct activity changes.
- Further research is needed to elucidate the intricate mechanisms governing Jun's transcriptional regulation.