Phosphorylation of transcription factors as a signal transduction mechanism in the archaeon Sulfolobus acidocaldarius.

Hassan Ramadan Mohamed Ahmed Maklad, Eveline Peeters

Research output: Unpublished contribution to conferencePoster


Despite being ubiquitous and having unique and diverse metabolic capacities, Archaea constitute the least studied
domain of life. Archaea were classified as Bacteria for decades until Carl Woese and his co-workers proposed
a tripartite phylogenetic division composed of the domains Bacteria, Archaea and Eucarya.
Today, the existence of reversible protein phosphorylation in the three domains of life is well established and it
is thoroughly studied in Eucarya and Bacteria. However, in Archaea little is still known about protein
phosphorylation regarding the target proteins, their cognate kinases/phosphatases and the regulatory processes
in which they are involved. As a model organism for the major archaeal family Crenarchaeota, S. acidocaldarius
gains a special interest to understand signal transduction cascades and the regulatory mechanisms involved in
them. Recently, a phosphoproteome study of S. acidocaldarius was done revealing many of the phosphorylation
target proteins and sites and characterizing the role of two identified protein phosphatases; Saci_PTP and
Saci_PP2A. It showed that in S. acidocaldarius 801 unique proteins were found to be phosphorylated in vivo.
The phosphorylation occurs on serine, threonine and tyrosine residues and intriguingly, tyrosine was the most
targeted residue.
The transcription factor FadRSa (a TetR-like transcription factor encoded by Saci_1107that regulates a gene
cluster involved in fatty acid metabolism.) was found to be among the phosphorylated transcription factors in
vivo. The detected phosphorylation is occurring on the three residues Y133, T134 and T135. FadRSa is
We investigated the phosphorylation event occurring in the ligand binding pocket of FadRSa regarding the protein
kinases involved and the possible outcome on DNA binding in vitro as a case study of phosphorylation
regulatory function in Achaea.
Using the bioinformatics tools Archaeal Clusters of Orthologous Genes browser (arCOGs) and Archaeal and
Bacterial Synteny Explorer (Absynte) we were able to identify 12 putative protein kinase genes in the genome
of S. acidocaldarius. The putative genes were cloned in pET28b vector with N-terminal 6x Histag. Six of them
were successfully expressed in E.coli RosettaTM(DE3) strain and the proteins were purified on His-trap column
using ÄKTA FPLC system.
In vitro phosphorylation assays using [γ32 P]-ATP revealed that FadRSa is specifically phosphorylated by the
eukaryotic type-like kinases ArnC and Saci1041. Mass spectrometry analysis showed that this phosphorylation
occurs only on threonine and serine residues, thus evoking the question which kinase is responsible for the
tyrosine phosphorylation. Using bioinformatics tools we screened S. acidocaldarius kinase sequences and were
able to identify sequence similarities between the putative kinases Saci1289 and Saci2317 and bacterial-type
tyrosine kinase motifs. Protein purification and phosphorylation assays of both kinases are currently ongoing .
In vitro, the binding of the specific acyl-CoA ligand in the FadRSa ligand binding pocket causes disruption of
FadRSa-DNA complexes in electrophoretic mobility shift assays. However, when performing the same assay with
a tyrosine phosphomimetic FadRSa mutant, the protein became clearly less responsive to ligand binding(Figure
2). Thus, the tyrosine phosphorylation of FadRSa in its ligand binding pocket is postulated to have a regulatory
role in response to the energetic status of the cell.
Original languageEnglish
Publication statusPublished - 19 Oct 2018
EventAnnual Symposium for the Belgian Society of Microbiology -
Duration: 19 Oct 201819 Oct 2018


ConferenceAnnual Symposium for the Belgian Society of Microbiology


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