ERK-mediated phosphorylation of fibroblast growth factor receptor 1 on Ser777 inhibits signaling.

Fibroblast growth factor 1 (FGF1) controls cellular activities through the activation of specific cell-surface FGF receptors (FGFRs). Transphosphorylation of tyrosine residues in the kinase domain of FGFRs leads to activation of intracellular signaling cascades, including those mediated by mitogen-activated protein kinases (MAPKs). FGFRs also contain a serine-rich C-terminal tail. We identified a regulatory mechanism of FGFR signaling involving phosphorylation of Ser(777) in the C-terminal region of FGFR1 by the MAPKs extracellular signal-regulated kinase 1 (ERK1) and ERK2. Prevention of the phosphorylation of Ser(777) in FGFR1 or mutation of Ser(777) to alanine enhanced FGF-stimulated receptor tyrosine phosphorylation and increased cell proliferation, cell migration, and axonal growth. A form of FGFR1 with a phosphomimetic mutation at Ser(777) exhibited reduced signaling. Activation of MAPKs by other receptor tyrosine kinases also resulted in phosphorylation of Ser(777) in FGFR1, thereby enabling crosstalk regulation of FGFR activity by other signaling pathways. Our data reveal a negative feedback mechanism that controls FGF signaling and thereby protects the cell from excessive activation of FGFR.

Replication of plasmids derived from Shiga toxin-converting bacteriophages in starved Escherichia coli.

The pathogenicity of Shiga toxin-producing Escherichia coli (STEC) depends on the expression of stx genes that are located on lambdoid prophages. Effective toxin production occurs only after prophage induction, and one may presume that replication of the phage genome is important for an increase in the dosage of stx genes, positively influencing their expression. We investigated the replication of plasmids derived from Shiga toxin (Stx)-converting bacteriophages in starved E. coli cells, as starvation conditions may be common in the intestine of infected humans. We found that, unlike plasmids derived from bacteriophage λ, the Shiga toxin phage-derived replicons did not replicate in amino acid-starved relA(+) and relA(-) cells (showing the stringent and relaxed responses to starvation, respectively). The presence of the stable fraction of the replication initiator O protein was detected in all tested replicons. However, while ppGpp, the stringent response effector, inhibited the activities of the λ P(R) promoter and its homologues from Shiga toxin-converting bacteriophages, these promoters, except for λ P(R), were only weakly stimulated by the DksA protein. We suggest that this less efficient (relative to λ) positive regulation of transcription responsible for transcriptional activation of the origin contributes to the inhibition of DNA replication initiation of Shiga toxin-converting bacteriophages in starved host cells, even in the absence of ppGpp (as in starved relA(-) hosts). Possible clinical implications of these results are discussed.

Transcription regulation of the Escherichia coli pcnB gene coding for poly(A) polymerase I: roles of ppGpp, DksA and sigma factors.

Poly(A) polymerase I (PAP I), encoded by the pcnB gene, is a major enzyme responsible for RNA polyadenylation in Escherichia coli, a process involved in the global control of gene expression in this bacterium through influencing the rate of transcript degradation. Recent studies have suggested a complicated regulation of pcnB expression, including a complex promoter region, a control at the level of translation initiation and dependence on bacterial growth rate. In this report, studies on transcription regulation of the pcnB gene are described. Results of in vivo and in vitro experiments indicated that (a) there are three σ(70)-dependent (p1, pB, and p2) and two σ(S)-dependent (pS1 and pS2) promoters of the pcnB gene, (b) guanosine tetraphosphate (ppGpp) and DksA directly inhibit transcription from pB, pS1 and pS2, and (c) pB activity is drastically impaired at the stationary phase of growth. These results indicate that regulation of the pcnB gene transcription is a complex process, which involves several factors acting to ensure precise control of PAP I production. Moreover, inhibition of activities of pS1 and pS2 by ppGpp and DksA suggests that regulation of transcription from promoters requiring alternative σ factors by these effectors of the stringent response might occur according to both passive and active models.

The P1 promoter of the Escherichia coli rpoH gene is utilized by sigma 70 -RNAP or sigma s -RNAP depending on growth phase.

The P1 promoter of the Escherichia coli rpoH gene has been known as a sigma(70)-dependent promoter (RNAP). We show here that it is also recognized by sigma(S). The sigma(70) and sigma(S) RNA polymerase subunits direct transcription from the P1 promoter in the exponential and stationary growth phases, respectively, and transcriptional start sites for the two holoenzymes differ by 1 nt. The transcription after heat shock is sigma(70)-dependent. The results are based on (1) sequence analysis that revealed features of promoters responsive to both, sigma(70)- and sigma(S)-RNAP, (2) in vitro transcription experiments verifying that both holoenzymes are able to transcribe the promoter, (3) electron microscopy results indicating that both holoenzymes bind the same site, (4) primer extension test performed with RNA isolated from the wild-type and rpoS mutant strains, demonstrating that transcription from the P1 promoter in the stationary phase is sigma(S)-dependent. These and previous results point to cooperation of sigma(70), sigma(24), sigma(S) and sigma(54) regulons in the expression of the rpoH gene, coding for the main regulator of the stress response, thus rendering it active in a variety of conditions.