Peer review of: The receptor PTPRU is a redox sensitive pseudophosphatase (2019)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

NCOMMS-19-37816 

The manuscript by Hay et al describes the structure and characterization of the PTPRU 
pseudophosphatase, a receptor tyrosine phosphatase that is proposed to be proto-oncogenic, 
although its true function is still not fully established. The biological function of this protein is the 
question addressed in this manuscript, which describes biochemical, structural and cell biological 
data unified by an effort to uncover its true function in cells. The experiments are solid and 
interesting, providing new intriguing possibilities about the role of this protein in biology and cancer. 
That being said, some of the conclusions lack the solidity one might expect from a Nature 
Communications paper and thus a few additional studies are proposed that are designed to 
strengthen the manuscript conclusions, especially regarding mechanism. Unraveling these questions 
are very important to the field of phosphorylation signaling. 

 

1.Activity assays: The enzyme activity assays are solid and convincing. However, no rationale is 
provided as to why PTPRK was selected for the comparative studies and a sentence or two at the 
beginning of this paragraph would strengthen this entire section (it took a couple of looks at the 
figure to realize some of the data refer to PTPRK). If the PTP loop Cys is replaced by Ser, are there 
any phenotypes? Similarly, is it perfectly conserved across all organisms that contain PTPRU genes? 
This might also provide insights into the role of this protein (and confirm lack of activity is ‘fine’). 

 

2.Structure: This section is very interesting, but a few additional experiments would strengthen the 
conclusions. The loss of an organized pTYR substrate specificity loop could certainly impact activity 
[although, this loop is often expected to restrict substrates to pTYR and one *might* predict that its 
absence could render the protein more active and certainly to more substrates, ie pS or pT]. Further, 
the authors discover an unusual conformation for the PTP loop originally hypothesized (but later 
proven by the authors to be the sole cause of PTPRU inactivity) that this is due to the presence of a 
Thr residue when its normally Ala, whose conformation also restricts pY access. To solidify the 
conclusions, the following experiments are suggested: 

 

a.CHIMERA: Make a chimera to test the pTyr substrate recognition loop hypothesis: replace the 
PTPRU residues 904-925 with the corresponding residues from PTPRK and visa versa. Test activity. 
Additional structures (if possible) would make this section stronger but crystallography is never 
predictable and thus not required. But activity assays are absolutely necessary. For the chimera’s, 
also perform the proteolytic cleavage and stability assays. This should provide strong confirmation if 
the author hypothesis is true. 

 

b. If the PTPRU-PTPRK904-925 (these exact residue numbers are likely different as these are the 
PTPRU numbers) chimera is not active, add the T->A mutation in the PTP loop as well (and, visa versa 
for the PTPRK-PTPRU(904-925)). Test activity. 

 

c. Together, these experiments will provide the data needed to conclude that these two 
sequence/structural differences are what renders PTPRU inactive. 

 

RMSD: 1.014—too many significant digits (for the accuracy of crystal structures); 1.0 will suffice. 

 

The crystallization pH is unusually high for PTPs. If another crystal form appeared, one might collect 
to determine if the structure is the same across different pH values. 

 

See also the comment in the figure—namely that from the figure 3A there *seems* to be a second 
PTPRU loop that may be the cause of the unusual PTP loop conformation. If this second loop does, in 
fact, overlap with the ‘typical’ position for the residue that corresponds to the Thr of the PTP loop, 
additional experiments to test this are warranted. 

 

 

3. Oxidation. While interesting, the biological importance is unclear (since it is not an active 
phosphatase). Based on the data presented, one might ask, would abolishing the ability to form a 
backdoor disulfide impact PTPRU function? i.e., if the backdoor cys was mutated to ser, would there 
be any phenotypes? 

 

Figure edits. 

 

Figure 1: 

A. This would benefit with something that ‘highlights’ the members of this family (M/K/T/U)—is 
there anything different about them that one might be able to add to this figure? Maybe, difference 
in activity; something else? Why are the D domains different colors—does this have an implied 
meaning (if it does, it is unclear what it is; green/red are mentally correlated with ‘go’/’stop’ and one 
might pick different colors if this is not what is implied). 

 

B. Since the manuscript (and experiments) are focused on ‘U’ and ‘K’, put these at the top of the 
alignments, not the bottom, with ‘U’ at the top. 

 

B-G. Exploit color. Use a different color for the font for PTPRK and PTPRU text (on gels, kinetic 
assays, ets) for all labels so its easier for readers to ‘distinguish’ the data corresponding to different 
constructs—these names are too similar for the non-experts and it takes a few looks before one 
realizes the constructs differ—make it obvious. 

 

Figure 2: 

A and B do not really illustrate what the authors are trying to show. For example, it is impossible to 
‘see’ where the disordered loop ‘ends’ are in PTPRU (they are not even labeled). Instead, show an 
example of how the pY SRL restricts access to only pY residues (using one of the PTP structures with 
a pY at the active site). Only real experts understand the importance of this loop without a figure-- 
give the broad audience of NC the chance to understand the argument. 

 

With the overlay in A, also show each structure separately (this will make the ‘missing’ loop more 
obvious). In the overlay, also make the pY loop (missing residues) light grey in the PTPRK structure; 
visually, this will be easier for the reader to ‘see’ what is missing. 

 

B is much less useful and might be fine for supplemental. Alternatively, just the overlaid pY loops as 
an ‘inset’ somewhere will illustrate the point the authors are making. Also, for this overlay, one 
might try ribbon instead of cartoon in pymol (some of the secondary structural elements are not 
uniformly defined among all structures—they likely overlay really perfectly with ribbon). 

 

C. put a triangle indicating increasing concentrations of trypsin over the numbers—visually easier. 
[Some of this figure may change based on the recommended chimera experiments.] 

 

 

Figure 3. 

A. State at least the number of PDBs overlaid in the legend. Label T1089. Also, it is clear from ii and iii 
in this panel that there is another PTPRU loop that ‘appears’ to overlap the residues that correspond 
to the ‘T’ in PTPRU (looks like alanines and some other amino acids)—that is, is this OTHER loop the 
reason the PTP loop moves? i.e., because there is a second loop in PTPRU to pushes is up out of the 
way. It is a little hard to see here but this might explain why the PTP loop is in such an unusual 
conformation (if it actually does clash with the ‘canonical position for the Thr residue sidechain). 

 

C. This figure is not useful as it appears that it is trying to make the same point as panel iii in ‘A’. 
Delete it. 

 

Figure 4: move to supplemental? Its simply unclear if this ability of PTPRU has any biological 
relevance and thus it may be more artifactual (not lost in evolution) than anything else. 

 

Fig 5, A. The different colors are very helpful. Indicating which domains are active on this figure 
would be extremely helpful for more easily interpreting the rest of this figure. 

English: Reread for English. There are a handful of typos and awkward sentences. 

 

 

 

Reviewer #2 (Remarks to the Author): 

 

The receptor PTPRU is a redox sensitive pseudophosphatase 

 

Hay …… Deane 

 

NCOMMS-19-37816 

 

The authors present a detailed biochemical analysis of PTPRU. Using a variety of approaches, they 
have not been able to demonstrate phosphatase activity in their preparations of PTPRU. On this 
basis, they conclude that PTPRU is catalytically inactive. They present a crystal structure of the 
membrane-proximal, D1, domain of PTPRU and note some structural anomalies compared to known 
catalytically-competent PTP domains. They note oxidation of the “catalytic” Cys residue in PTPRU D1 
and engagement of this Cys by a “back-door” Cys residue in a disulfide bond, as has been reported 
for other PTPs. They also present domain swapping studies and suggest that the data should be 
interpreted by a mechanism of action in which “PTPRU can bind substrates and protect them from 
dephosphorylation by related phosphatases”. Overall, the studies are thorough and well-performed. 
Nevertheless, I feel like this is Bertrand Russell’s Teapot territory. The authors are trying to prove a 
negative – that a protein that looks like a member of the PTP family does not possess intrinsic 
catalytic activity. This is a difficult task but, as with the teapot, I think the burden of proof lies with 

them to demonstrate conclusively that PTPRU is not a phosphatase. In this case, I do not think they 
have done so satisfactorily. What is left is a series of biochemical observations that are not unique 
and have been reported already for other members of the PTP family; as such, I do not think this 
rises to the level required for Nature Communications. 

 

I think the pseudoenzyme field is a fascinating area of research. Unfortunately, it is replete with 
examples of proteins that were classified originally as inactive pseudoenzymes, but with further 
analysis were shown actually to be enzymatically active. The kinases provide a number of such 
examples – including HER3, KSR, JH2 domain in JAK etc. The protein tyrosine phosphatases (PTPs) 
themselves include several such cases. It is interesting to note that the authors, in their Introduction 
on page 3, highlighted PTPN23 (HD-PTP), PTPN14 (PTPD2) and PTPN21 (PTPD1) as being catalytically 
inactive. Nevertheless, there are papers published, by several different labs, attesting to the fact that 
these PTPs do possess intrinsic activity. The authors should have presented a more thorough and 
balanced introduction. Furthermore, this suggests that great caution should be exercised before 
concluding from limited biochemical analysis that a given protein is a pseudoenzyme – it may just be 
that you have yet to test the correct substrate. 

 

The authors place emphasis on the observation that several important sequence motifs in PTPRU 
have diverged from the canonical motifs observed in catalytically competent PTPs. In particular, 
“PTPRU possesses non-canonical WPD (WPE) and pTyr recognition loop sequences (GSRQ rather 
than KNRY), as well as a unique threonine in the PTP loop”. Nevertheless, it is important to note that 
other PTPs, such as PTPRQ and PTPN23, also display a WPE motif, but still possess intrinsic activity. 
Even in the study of PTP1B cited by the authors, it was reported that although mutation of the D in 
the WPD loop was inhibitory, the mutant protein maintained a measurable level of activity. This 
raises the question, just how much activity against an artificial substrate in vitro is necessary to 
underlie a physiological function? It should be noted that like PTPRU, PTPN23 also presents a non-
canonical pTyr recognition loop. Finally, the authors themselves report here that “introduction of a 
threonine to the active site was not sufficient to inactivate PTPRK D1” (see page 6) and, therefore, 
this Thr in PTPRU is unlikely to be the cause of their failure to demonstrate activity. Consequently, I 
think one should be cautious about placing too much reliance on such sequence analyses. 

 

The data presented in this report suggest that PTPRU was a difficult PTP to work with. For example, 
on page 7, the authors state that “we were unable to purify full PTPRU-ICD (D1 +D2)” and they were 
unable to generate chimeras containing PTPRU D2. Most of their analysis of potential activity in 
PTPRU was conducted on a truncated construct expressing the isolated D1 domain as a recombinant 
protein in E. coli. How do they know that the protein folded properly? There have been reports to 
suggest a chaperone function for D2 domains – perhaps the presence of D2 is essential for 
manifestation of catalytic activity in PTPRU? Also, it was reported by Philip Cohen’s lab that, at least 
for protein phosphatase 1, the activity of the protein expressed in E. coli possessed different 
features from the activity of the phosphatase purified from mammalian tissues [MacKintosh et al 

(1996) FEBS Letts 397: 235-238]. With this in mind, the presence of disordered segments in the 
structure of the recombinant protein may reflect incorrect folding in E. coli. 

 

 

 

Reviewer #3 (Remarks to the Author): 

 

In the manuscript, “The receptor PTPRU is a redox sensitive pseudophosphatase,” Hay et al provide 
evidence that the protein tyrosine phosphatase D1 domain (PTP D1) of the receptor phosphatase 
PTPRU lacks catalytic activity. The authors also show that the protein’s D1 domain is less 
thermostable than a homologous PTP domain and they solve the first crystal structure of PTPRU’s 
PTP D1 domain, uncovering deviations from canonical PTP-domain structure that are consistent with 
PTPRU D1’s lack of activity and instability. Finally, the authors provide preliminary evidence that that 
the intracellular portion of PTPRU can associate with substrates of other receptor PTPs (e.g., p120-
catenin), sequestering them from potential dephosphorylation; when PTPRU is knocked out, the 
tyrosine phosphorylation of p120-catenin counterintuitively decreases. 

 

 

Receptor PTPs are key regulators of cell signaling, and the demonstration that a member of the 
family is not really a PTP (i.e., a pseudophosphatase) would represent a significant finding. The 
manuscript is well written and the experiments are well designed and clearly presented. However, a 
revised version of the manuscript would need to address the following critiques before I could 
recommend it for publication. 

 

Critique 1: Cropping of anti-phosphotyrosine westerns (Figures 1E, 1G, and 5E). The anti-pTyr blots 
purport to demonstrate the lack of activity of the PTPRU D1 against a whole range of potential 
substrates. So, why are the blots cropped at seemingly arbitrary molecular weight cut-offs? The 
authors should present the uncropped blots to show the tyrosine-phosphorylation levels over the 
entire molecular-weight range of proteins in the treated lysates. 

 

Critique 2: How have the authors controlled for the possibility that the lack of activity in their PTPRU 
D1 constructs may be an artifact of truncating the protein? The thermal instability of the protein—as 
well as the mysterious inability to express the D1+D2 construct—suggest that this protein doesn’t 
“like” to be truncated. The authors should attempt experiments in which they assess activity in the 
context of the full-length protein. Perhaps immunoprecipitating PTPRU side-by-side with another 
RPTP and then comparing the PTP activities in the IPs? 

 

Critique 3: The authors should more fully engage the previous literature that suggests that PTPRU is 
an active phosphatase. The authors state only in passing in the introduction that “… previous work 
suggests that PTPRU can dephosphorylate substrates such as β-catenin.” Indeed, one of the papers 
that the manuscript references (Yan et al, 2006) shows rather convincing evidence that expression of 
PTPRU causes a dramatic decrease in phosphorylation of β-catenin’s Y102, and that this decrease is 
dependent on the presence of catalytically active PTPRU (the dephosphorylation is completely lost 
when an inactive Cys to Ser mutant is expressed instead of the wild-type protein). How do the 
authors square this previous finding with the lack of PTP activity in their constructs? Have they 
attempted to look at PTPRU’s activity with this previously reported substrate?