Evidence for phosphatase activity of p27SJ and its impact on the cell cycle
Authors: Armine Darbinyan
Surekha Bonasu
Eric Chabriere
Kamel Khalili
Marta Czernik
Shohreh Amini
Nune Darbinian
Mikael Elias
Journal: Journal of Cellular Biochemistry (2009)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: p27SJ; DING proteins; phosphatase activity; Erk1/2; signaling pathways
Affiliations: Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, Philadelphia, Pennsylvania 19122, USA.

Abstract:  p27SJ, a novel protein isolated from St John's wort (Hypericum perforatum), belongs to an emerging family of DING proteins that are related to a prokaryotic phosphate-binding protein superfamily. Here we demonstrate that p27SJ exhibits phosphatase activity and that its expression in cells decreases the level of phosphorylated Erk1/2, a key protein of several signaling pathways. Treatment of p27SJ-expressing cells with phosphatase inhibitors including okadaic acid, maintained Erk1/2 in its phosphorylated form, suggesting that dephosphorylation of Erk1/2 is mediated by p27SJ. Further, expression of p27SJ affects Erk1/2 downstream regulatory targets such as STAT3 and CREB. Moreover, the level of expression of cyclin A that associates with active ERK1/2 and is regulated by CREB, was modestly reduced in p27SJ-expressing cells. Accordingly, results from in vitro kinase assays revealed a noticeable decrease in the activity of cyclin A in cells expressing p27SJ. Cell cycle analysis demonstrated dysregulation at S and G2/M phases in cells expressing p27SJ, supporting the notion that a decline in cyclin A activity by p27SJ has a biological impact on cell growth. These observations provide evidence that p27SJ alters the state of Erk1/2 phosphorylation, and impacts several biological events associated with cell growth and function. 

Retracted: No

PubPeer comments:
 Figure 4C. * Red boxes: The 32h panels of the -Dox and +Dox group appear to look unexpectedly similar. Yet, the cell cycle percentages are different. * The 0h panels also appear to look identical, with identical percentages, but that could mean Dox was added immediately after the measurement - although the experiment was done in duplicate so another panel might have been available. Could the authors please check? ![file] , 
 One of the authors pointed out on twitter that there are very small differences between the two 32 hour panels. However, it seems quite possible that these differences are simply compression artifacts due to the low image quality. It seems very unlikely that two different experimental conditions would yield such similar graphs. The raw data would be helpful to clarify this issue. , 
 I realize that I had responded to Dr. Bik on her twitter comment, but not here. I am only a minor contributor of this manuscript (Fig.2), and not an expert in flow cytometry. I have been communicating with the first author on this point. 1.tI will not comment on the 0 hr time. The fact that it is even mentioned here demonstrate you have not read understood the paper. 2.tFor the 32 hours, in accordance with discussions with the first author, extremely close graphs are expected and consistent with how close cell distributions are 3.tThe graphs do not appear identical when zoomed ![file] 4.tThe first author is still looking for higher resolution pictures original data to help clarify this. Yet, the age of the paper, past lab moving and the COVID situation has slowed this considerably. 5.t2nd comment, anonymous: I was told that pubpeer had strict moderation policies in regards to pointless comments. , 
 Thanks for providing this. This is indeed intriguingly similar. We will be updating this thread when Dr. Darbinian finds the original data. , 
 #3 If I may join in with this interesting discussion, the best resolution image for Fig. 4C in the public domain appears to be linked from the NIH PMC page https: www.ncbi.nlm.nih.gov pmc articles PMC2769080 Colourising the top right FACS image black pixels to red and the lower left FACS image to blue and then making the red image 50% transparent allows us to superpose the two plots and eyeball them for degree of overlap. They look to be exactly the same images to me. Apart from the labelled cell cycle percentages, that is. Personally speaking, Condy doesnu2019t need the authors to find a higher resolution image than that supplied at PMC. ![image] , 
 We are contacting the first author who was the individual who performed most of the experiments. Will update accordingly. , 
 #1 Hi, We really appreciate your interest to our manuscript and questions you are raising. This particular experiment was done by reading each sample by flow cytometer at FACS facility and results were generated by the instrument and sent to us. Software of the flow cytometer used numerical values obtained by the flow cytometer during analysis of each individual sample to create graphs plots corresponding to each sample. Cells were harvested at each time point, fixed and fed to the machine. Each sample can enter the machine only once and only after reading of one sample the next sample can be read (you can see that original files have different dates time points: 1 30 2009 at 11:06 am and at 1 30 2009 at 11:09 am). Since no major changes were detected at 32 h between 2 samples, then no significant differences were recorded leading to absence of noticeable differences on the graphs at that particular time point of cell cycle. To add, main findings of that experiment were centered at 24 h and 28 h time points. I hope our explanations address your questions, but, please, feel free to email if you have more questions. I think this type of discussions could be very educational. Best, Nune , 
 Dear Nune, Thank you for your reply - much appreciated! However, I am not sure I can follow your explanation, and I do not think you addressed the similarities. With the interest of the reader in mind, I will continue the discussion here, instead of per email. First, you wrote about the timepoints of the original files, but I cannot see those original files. It would be helpful if you could upload the original files of all 10 panels somewhere (e.g. Dropbox, Google Drive) and post the link here. Sharing original data might take away any concerns, much better than explaining to me how flow cytometry works. Second, are you saying that these two plots marked by me with red boxes are indeed two different files? Or are they the same samples? I could not read a clear answer to that question in your reply. , 
 #7 To be frank, this response from the first author is nonsensical obfuscation. RE: * Since no major changes were detected at 32 h between 2 samples, then no significant differences were recorded leading to absence of noticeable differences on the graphs at that particular time point of cell cycle. * In the figure, the % values for the three cell cycle windows are clearly different between the two 32 hour samples, so how can this be reconciled with your claim that * no significant differences were recorded *??? And, FACS plots are never this identical between independent samples; it is obvious that the two sets of profiles are identical, not just similar. Why does the plainly obvious need to be explained repeatedly to the authors? , 
 Dear Dr. Bik and others, I will answer here as a minor author of this study to attempt to clarify Dr. Darbinianu2019s findings. We were alerted by Dr. Biku2019s comment on the similarity of the fig 4B 32 hrs panels. The finding by another user here of higher resolution figures made it clear that the graphs are too similar. Dr. Darbinian has been investigating in her data files, and she found the source files from the facility. Dr. Darbinian mentioned that this experiment was outsourced to a cell sorting facility. Here are the source images (8.32-.jpg and 9.32+.jpg) from the facility found by Dr. Darbinian: ![file] ![file] We examined these images and found that they are superposable. We are not sure what the cause is for this duplication from the cell sorting facility. This error may be a duplication from the operator, or instrument issue. It could or could not have affected the cell distribution % values for this time point. The analysis made in the paper, and the conclusions are in fact independent from this specific time point (32hrs). The main conclusion from figure 4C is that cell cycle (S and G2 M phases) is being dysregulated in cells expressing p27SJ. In fact, this is very clearly observed in at times 16hrs, 24hrs, 28hrs, but only very weakly at 32hrs (looking at distribution % values). If anything, the cell phases distribution for this time point makes the figure conclusion weaker. I hope this helps clarify things. The journal editor will now be contacted to discuss the matter and resolve it. Thank you for your help in identifying this error. , 
 Below are the other images found by Dr. Darbinian: 1.0h.jpg ![file] 2.16-.jpg ![file] 3.16+.jpg ![file] 4.24-.jpg ![file] 5.24+.jpg ![file] 6.28-.jpg ![file] , 
 7.28+.jpg ![file] , 
 Thank you, Dr. Elias for posting these original images. That is very helpful. It appears that the two panels marked in comment #1 have indeed been duplicated, although it might be hard to reconstruct how this has happened. I agree that this most likely looks like an honest error, and it would be best addressed with an erratum. Thank you and Dr Darbinian for finding and posting these files. , 
 #13 Thank you, Dr. Bik, We are happy we found original old files that provide more information. Nune , 
 Erratum, October 2021, found here: https: onlinelibrary.wiley.com doi 10.1002 jcb.30115  Unfortunately, in the article u201cEvidence for phosphatase activity of p27SJ and its impact on the cell cycleu201d by Nune et al. published in the Journal of Cellular Biochemistry 107:400-407 (2009),1 Figure 4, its legend and the corresponding text had errors. The revised figure and the text describing Figure 4C on page 404 are as follows. The correct text describing Figure 4C, section u201cResults and Discussion,u201d p. 404 should read: As seen in Figure 4C, In the absence of p27SJ (no doxycycline treatment), cells progressed from G0 G1 to S at 24u2009h and to G2 M at 28u2009h after release from serum starvation. In cells expressing p27SJ, cells also progressed from G0 G1 to S phase at 24u2009h with approximately 8% decreased levels compared with those from p27SJ-negative cells. Also, progression of the cells from S to G2 M was reduced in p27SJ cells. Unlike the control cells, which had 27.4% of the cells at G2 M stage, only 16.5% of the cells expressing p27SJ at 28u2009h were detected at the G2 M phase. These results taken together show that dysregulation of cell cycle factors including cyclin A, which was shown to impact S and G2 phases (Pagano et al., 1992), has a biological consequence on cell cycle progression of the p27SJ-expressing cells. New Figure 4: ![file] The Erratum does not specify what the errors were, but it corrects the duplication in the 32h panels from Figure 4C simply by removing the duplicated panels. Very creative! ,