The MDM2 ubiquitination signal in the DNA-binding domain of p53 forms a docking site for calcium calmodulin kinase superfamily members
Authors: Ted R Hupp
Nathalie Sphyris
Ben J Harrison
Irena Dornreiter
Jennifer A Chrystal
Yao Lin
Mary T Scott
Ashley L Craig
Jennifer A Fraser
Journal: Molecular and cellular biology (2007)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: p53; Ser(20) phosphorylation; CHK1; DAPK-1; calcium calmodulin kinase superfamily
Affiliations: University of Edinburgh, Cancer Research Centre, Edinburgh EH4 2XR, UK., University of Edinburgh, Cancer Research Centre, CRUK p53 Signal Transduction Group, South Crewe Road, Edinburgh, United Kingdom EH4 2XR, Heinrich-Pette-Institut fur Experimentelle Virologie und Immunologie, Universitat Hamburg, Hamburg, Germany

Abstract:  Genetic and biochemical studies have shown that Ser(20) phosphorylation in the transactivation domain of p53 mediates p300-catalyzed DNA-dependent p53 acetylation and B-cell tumor suppression. However, the protein kinases that mediate this modification are not well defined. A cell-free Ser(20) phosphorylation site assay was used to identify a broad range of calcium calmodulin kinase superfamily members, including CHK2, CHK1, DAPK-1, DAPK-3, DRAK-1, and AMPK, as Ser(20) kinases. Phosphorylation of a p53 transactivation domain fragment at Ser(20) by these enzymes in vitro can be mediated in trans by a docking site peptide derived from the BOX-V domain of p53, which also harbors the ubiquitin signal for MDM2. Evaluation of these calcium calmodulin kinase superfamily members as candidate Ser(20) kinases in vivo has shown that only CHK1 or DAPK-1 can stimulate p53 transactivation and induce Ser(20) phosphorylation of p53. Using CHK1 as a prototypical in vivo Ser(20) kinase, we demonstrate that (i) CHK1 protein depletion using small interfering RNA can attenuate p53 phosphorylation at Ser(20), (ii) an enhanced green fluorescent protein (EGFP)-BOX-V fusion peptide can attenuate Ser(20) phosphorylation of p53 in vivo, (iii) the EGFP-BOX-V fusion peptide can selectively bind to CHK1 in vivo, and (iv) the Deltap53 spliced variant lacking the BOX-V motif is refractory to Ser(20) phosphorylation by CHK1. These data indicate that the BOX-V motif of p53 has evolved the capacity to bind to enzymes that mediate either p53 phosphorylation or ubiquitination, thus controlling the specific activity of p53 as a transcription factor. 

Retracted: No

PubPeer comments:
 Figure 6F panel appears to be composite, whereas the others don t. ![file] , 
 Figure 7. P and Q much more similar than expected. ![file] , 
 #1 Figure 6. Higher resolution. Vertical, straight changes in signal in panel F, but not in the other panels. ![file] , 
 Query 1. Figure 7P and 7Q The authors agree that these data appear similar. The original blots are no longer available for resolving this query so the original data from the paper was downloaded from the journal web site. The purpose of Figure 7 P-S was to measure Enhanced green Fluorescent Protein (EGFP) in human cells after transfection. There can be no EGFP protein in blots P and Q because the EGFP was not transfected in (as in R and S), so the absence of any immuno-bands is consistent with lack of EGFP. We think the similarities between 7P and 7Q such as in the white dots (circled in blue and red) are due to u2018self-glowu2019 plastic orientation markers that we used at the time to place at the bottom of the X-ray film cassette to orientate the bottom and top of blots when x-ray film is placed in the cassette. The similarity in the black dot in green could come from contaminants in the X-ray film cassette. Nevertheless, despite these similarities, there are differences between the two blots (image below), in terms of horizonal streaks highlighted at different positions. The differences in the horizonatal streaks on the two blots likely come from abrasions in the developer wheels that scratch the surface of the film and create horizontal streaks. Since these streaks are located in different positions, we think these blots are not identical. https: pubpeer.com storage image-1642075597394.png Query 2. Figure 6F. We agree that Figure 6F appears spliced. The original image is no longer available, but it is likely the image was spliced from a larger gel (that was not loaded in the same sequential manner) in order to collate and visually orientate lanes 3 and 4 to other lanes 3 and 4 in the Figure. That is to say, lanes 3 and 4 are the key elements of the Figure which compares 6F and 6H. In which panel 6F, lanes 3 and 4, shows the effects of CHk1 transfection on p53 phosphorylation whilst Panel 6H, lanes 3 and 4, shows the effects of DAPK transfection on phosphorylation of p53. Since lanes 3 and 4 from figure 6F were spliced into this position of the figure for orientation purposes, this does not effect the conclusion of the experiment. In addition, the other controls include lanes 5 and 6 (6F and 6H) in which there is no phosphorylation detected by DAPK or CHK1 on the p53-deletion protein. ,