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Poster Szbk17 Aladar
Poster Szbk17 Aladar
a global repressor and Myb transcription factors counteract the repression (1). Although plants share the conserved A B C 1/50
N T N T
members of E2F, DP, RB, Myb proteins (2;3), and some MuvB components, the existence of DREAM repressor complex
and its possible functions during cell proliferations are not known. By using various biochemical approaches, such as P-RBR
The E2FB transcription factor is not just nuclear localised but seems to present in the plasma
membrane. E2FB interacts with PIN proteins
A B
PIN3-GFP
PIN3-GFP
p35S-GFP
Input 1/50
IP-GFP
IP-GFP
E2FB
pE2FA:gE2FA-3xvYFP Loading
control
GFP
(ponceau)
pE2FB:gE2FB-3xvYFP
Cell cycle activator E2FB transcription factor was detected in cell boundaries of transgenic Arabidopsis leaf expressing E2FB in
fusion with triple Venus-tag (3xvYFP) (green signal) under the control of its own promoter. In contrast, closest relative E2FA
was predominantly nuclear localised in pE2FA:gE2FA-3xvYFP transgenic leaf. Confocal laser microscopy images were taken
after propidium iodide staining (red signal)(A). PIN3-GFP expressing plants were used to immunoprecipitate (IP) protein
complexes through the GFP epitope, and the presence of E2FB was detected by using E2FB specific antibody in immunoblott
assay(B).
Phospho@9
MYB3R3 and MYB3R4 both interact with RBR1 and differentially associate with E2F isoforms
Phospho@374|375
Phospho@385
A, B MYB3R3‐GFP and GFP‐MYB3R4 both interact with RBR1 and CDKA;1, but with a different E2F isoform in Arabidopsis leaves. Phospho@389
IP was performed with anti‐GFP antibodies from protein extracts prepared from first leaf pairs of MYB3R3‐GFP or GFP‐MYB3R4 Phospho@406
transgenic plants at indicated days after germination (DAG). In these transgenic plants, expression of GFP fusion proteins was Phospho@685
driven by the corresponding native promoters. Co‐IP of RBR1 and E2FB was examined by Western blot analyses using Phospho@708
corresponding antibodies. For detection of MYB3R3‐GFP and CDKA;1, anti‐GFP and anti‐PSTAIRE (specific to CDKA;1) Phospho@712|714
Phospho@885
antibodies were used. As input, 1/10 of IP was loaded. Coomassie staining of the same membrane was used as a loading control.
Phospho@898
Phospho@911
C. MYB3R3‐GFP interacts with E2FC, but GFP‐MYB3R4 does not. IP was performed with anti‐GFP antibodies from protein Phospho@936|937|940|942
extracts prepared from first leaf pairs of MYB3R3‐GFP or GFP‐MYB3R4 transgenic plants at indicated days after germination Phospho@940|942
(DAG). Co‐IP of E2FC and CDKA;1 was examined by Western blot analyses using anti‐E2FC and anti‐PSTAIRE antibodies, Phospho@936|942
respectively. As input, 1/16 of IP was loaded. Coomassie staining of the same membrane was used as a loading control. Phospho@942
Several phosphopeptides were identified by MS/MS from RBR1-GFP overexpressing plants, direct RBR1-GFP pull downs (RBR1
as bait) and from E2Fs-GFP pull downs (E2F bound, prey RBR1). Not surprisingly the phosphosites of the Arabidopsis RBR1 show
QUBIC workflow in label-free format strong similarities to the human ortholog. Interestingly, although the E2F bound RBR1 fraction contains several phosphosites,
the well studied S911( )phosphorylation, which is known to prevent E2F binding was present only in the free RBR1 fraction.
DREAM-complex interaction partners of E2FA, E2FB, E2FC, RBR1, DPA, DPB and MYB3R3
Seedlings (7 DAG) expressing GFP tagged E2FA/B/C, RBR1, DPA/B and MYB3R3 under their own promoters were collected and
Bolyai Research Scholarship of the Hungarian Academy of Sciences
used for separate immunoprecipitation experiments. Immuno Purified samples were analyzed by LC MS/MS Numbers indicate
given to A.P.‐Sz., Hungarian Scientific Research Found (OTKA
the number of identified unique peptides for the respective proteins. 105816) given to Z.M.
S4). Interestingly, in the E2FA-GFP pull downs we could never detect any of the components of the multi-protein DREAM The research was also supported by the Economic Development and
complex (DP, RB-like E2F, and MuvB, (1), while with E2FB-GFP, these proteins were readily Innovation Operation Programme (GINOP-2.3.2-15-2016-00032).
pull down. This may suggest that E2FA functions in different complex(es) (6) than the DREAM associated with E2FB and E2FC
(5). None of these proteins were identified when the GFP-expressing control plants were analyzed.