Alteration of a cyclic AMP-dependent protein kinase phosphorylation site in the c-Fos protein augments its transforming potential.

Abstract

We have studied the phosphorylation of the nuclear oncoprotein Fos by cyclic AMP-dependent protein kinase (PKA). We demonstrate that the human c-Fos protein, phosphorylated either in vitro with purified PKA 65 kDa (3,12). This heterogeneity is largely due to phosphorylation of serine residues (3). Although both c-Fos and its viral homolog v-Fos are phosphorylated, the cellular protein is more extensively phosphorylated (3). Major phosphorylation sites in c-Fos have been localized to its C terminus within the amino acid sequence that differs from that of v-Fos (3, 41). Precise identification of the serine residues phosphorylated, as well as the kinases involved in this phosphorylation, would help our understanding of the biological function of Fos phosphorylation.A large body of data implicates the involvement of the cyclic AMP (cAMP)-dependent protein kinase A (PKA) pathway in the regulation of both the fos gene and the Fos protein (31). Activation of the PKA pathway by adenyl cyclase agonists such as forskolin leads to Fos expression (2,7,8,21,26), presumably via interaction of the phosphorylated cAMP responsive element (CRE)-binding protein with the CRE in the fos promoter (25,36 PKA does phosphorylate the human c-Fos protein in vitro on a site also phosphorylated in vivo. We have identified serine residue 362 as the phosphorylated amino acid. Mutation of serine residues 362 to 364 to alanine enhances the transforming activity of c-Fos to levels comparable with those of v-Fos. It has previously been shown that lack of phosphorylation of c-Fos at its C terminus abolishes its ability to downregulate its own promoter but has no effect on transactivation of genes linked to an AP-1 site (29, 36). Our results suggest that part of the mechanism that leads to cell transformation by Fos might be attributed to the loss of a PKA phosphorylation site in v-Fos. Moreover, our results implicate PKA in regulation of Fos activity in a complex manner by controlling both induction and repression of the fos promoter. MATERIALS AND METHODSCell lines and culture conditions. The human choriocarcinoma cell line JEG3 (20)

Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase.

“…This domain (amino acids 361-380) has also been reported to be required for the transrepression activity (25,29,30) of c-Fos. These potential RSK and MAP kinase sites are separated by a single proline residue (Fig.…”

“…Upon serum stimulation, c-Fos is more extensively phosphorylated than v-Fos, due to this C-terminal region (23,(25)(26)(27)(28). Phosphorylation of the C terminus has been shown to be responsible for the transrepression activity (29,30), and truncation or mutations that block phosphorylation in this region enhance the transforming capacity of c-Fos (30).…”

“…Upon serum stimulation, c-Fos is more extensively phosphorylated than v-Fos, due to this C-terminal region (23,(25)(26)(27)(28). Phosphorylation of the C terminus has been shown to be responsible for the transrepression activity (29,30), and truncation or mutations that block phosphorylation in this region enhance the transforming capacity of c-Fos (30).c-fos gene induction correlates with the agonist-dependent activation of the mitogen-activated protein kinase (MAP kinase)/90-kDa ribosomal S6 kinase (RSK) signal transduction pathway (31-33). In addition, both kinases are nuclear and cytoplasmic (34), which may be a prerequisite for their participation in regulation ofgene expression.…”

Nuclear protein phosphorylation and growth control

“…However, c-Fos is more extensively phosphorylated than v-Fos (by about 2-3-fold) and contains unique sites of phosphorylation which lie close to the C-terminus, within the 40-amino-acid region of c-Fos which is substituted by other sequences in the v-Fos protein (Barber & Verma, 1987 (Ofir et al, 1990). In addition, mutation of residues 362-364 gives rise to a Fos protein which is activated for cellular transformation (Tratner et al, 1992) these kinases have been mapped (cdkl also phosphorylates the C-terminal transrepression domain), the specific residues which are phosphorylated are not known, nor is it yet certain whether these kinases phosphorylate physiological sites or affect Fos activity (Abate et al,199 lb). One particularly interesting observation is that Fos can be phosphorylated in vitro by doublestranded DNA-activated protein kinase (dsDNA-PK; LeesMiller & Anderson, 1991).…”

Conserved motifs in Fos and Jun define a new class of activation domain.

“…Regulating one of the activation modules (HOB1) would then leave the second module (HOB2) free to cooperate with a distinct module within the same protein such as that present at the carboxyl terminus. Interestingly, the carboxy-terminal domain of Fos that cooperates with HOB2 is phosphorylated by protein kinase A (Tratner et al 1992). Regulation by phosphorylation may turn out to be a common feature of activation modules that cooperate with HOB2.…”