PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Biochemistry and Chemical Biology (eLife)
Abstract
Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine protein phosphatase 2 (PP2A) holoenzyme, PP2A/B55α, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55α recruits p107, a pRB-related tumor suppressor and B55α substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615–626) encompassing the strongest p107 binding site. This enabled us to identify an ‘HxRVxxV 619-625 ’ short linear motif (SLiM) in p107 as necessary for B55α binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55α/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, ‘ p [ ST ]- P- x(4,10)-[ RK ]- V -x-x-[ VI ]- R .’ Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55α, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55α groove, and phosphosite presentation. Altogether, these data provide key insights into PP2A/B55α’s mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55α’s role in multiple cellular processes.
Article activity feed
-
-
This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on October 28 2020, follows.
Summary
PP1 and PP2A make up the majority of serine/threonine phosphatase activity in the cell. While substrate recognition has been studied for PP1 and PP2B, the substate recognition of PP2A holoenzymes are less understood. Here, Fowle et al. set to understand substrate recognition of B55/PP2A. Using a specific substrate of B55, p107, the authors identify a conserved binding motif (HxRVxxV) for recognition by B55 and show additional B55 substrates also contain this motif. This work incorporates many complementary structural and biochemical assays to delineate the binding and recognition of substrates by B55.
Essential Revisions
What is the evidence that this motif is only recognized by B5alpha/PP2A and not …
This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on October 28 2020, follows.
Summary
PP1 and PP2A make up the majority of serine/threonine phosphatase activity in the cell. While substrate recognition has been studied for PP1 and PP2B, the substate recognition of PP2A holoenzymes are less understood. Here, Fowle et al. set to understand substrate recognition of B55/PP2A. Using a specific substrate of B55, p107, the authors identify a conserved binding motif (HxRVxxV) for recognition by B55 and show additional B55 substrates also contain this motif. This work incorporates many complementary structural and biochemical assays to delineate the binding and recognition of substrates by B55.
Essential Revisions
What is the evidence that this motif is only recognized by B5alpha/PP2A and not other B55 family members? Are the residues identified in B5alpha critical to the p107 interaction (D197 and L225) conserved among all of the isoforms? If they are, can other B55 family members bind p107?
Have the authors looked for the HxRVxxV motif across the proteome? The author only state that they noticed that this motif was found in Tau, p130 and MAP2, but how many proteins contain these motifs? A list or understanding of the potential proteins which contain this motif could give researchers outside the field a link to understand the phosphatase important for their protein of interest.
For the P107 deletion mutants has the expression of each one been confirmed in Figure 1 and is decreased binding to PP2A B55alpha been normalized to the expression of these mutants.
Is the phosphorylation of p107 by CDK2 affecting the affinity of B55 binding to this substrate?
Have the authors considered measuring direct binding affinities using ITC/SPR for example to look at the effects of these various mutants in a cell free / in vitro system?
It would have been interesting to study the effects of the various B55 mutants on the endogenous phosphorylation of p107, Rb, and KSR?
To gain insights into the physiological role of the identified domain of p107 in PP2A-B55 binding and in the dephosphorylation of this protein, new "in cellulo" experiments using the full length p107 mutant protein have to be performed and its impact in the temporal pattern of dephosphorylation analyzed.
Figure 1D, it is obvious that in order to compare the levels of PP2A-B55 associated to each construct it is essential to normalize the levels of A and B55 signals to the quantity of protein that is recovered in each pulldown. As such, the levels of each GST construct in the pulldowns have to be measured by western blot and used to obtain the PP2A A our B55/GST-spacer ratio. Ratios can be then compared.
The authors state that: "a mutant lacking residues C-terminal of R2 binds B55α similarly to the full construct, indicating that residues C-terminal to the R2 domain are dispensable for B55α Binding". Do "residues C-terminal of R2" mean full R2 region? If this is the case, this statement is not supported by Supplementary Figure 1B, where western blot of construct 2 and 7 display dramatically reduced B55 and A levels.
The authors tested the effect of KR residue mutation in the R1 and R2 regions in p107 dephosphorylation. KR mutants used for the R1 are R621A/K623A, the two mutants that were tested in Figure 1D and that were shown to impact B55 binding. However, they select K657A/R659A for R2 region. These two mutants were not tested in Figure 1D. Why do they introduce these mutants and not R647A that was investigated in Figure 1D? If the authors think that these residues are important, why did they not test them for its capacity to bind B55 in Figure 1D?
Other cdk-dependent phosphorylation sites on p107 that are essential for E2F binding have been described. Some of these sites are out of the spacer sequence. It will be interesting to know whether the dephosphorylation of these sites are dependent on PP2A-B55 and regulated by the mutants on the spacer sequence that decrease B55 binding.
Figure 4A and B. Dephosphorylation pattern of R1R2 control construct is drastically different in Figure 4A compared to 4B. In the first case, complete dephosphorylation does only take place upon two hours of incubation compared with fifteen minutes in the second. This is very weird if the same purified phosphatase is used in both experiments. In this line, I would expect a timing of few minutes for a total dephosphorylation when a purified phosphatase is used. Does it mean that phosphatase in Figure 4A lost activity?
"In vivo" experiments on the dephosphorylation of the non-binding p107 full length mutants have not been performed. To demonstrate that these residues are physiologically relevant for the physiological temporal p107 dephosphorylation pattern, these experiments must be done.
In the same line, to really show the involvement of the pST-x(5-10)-(RK)-Vxx(VI)R in Tau dephosphorylation by PP2A-B55 a direct mutant of this sequence of Tau should be checked.
What are the consequences of B55a-interaction mutants in p107 function? Is that mutant protein able to sustain cell cycle arrest?
Since the authors propose a new model/motif, it would be great to add some statistics on to what extent this motif is present in the numerous hits found in recent screens for B55 targets during mitotic exit. Is this motif present in B55 targets involved in non-cell-cycle (TAU) or cell-cycle targets? Is it equally present in proteins dephosphorylated during early versus late mitotic exit? Any hint into these questions may facilitate the impact of the model proposed in the biology of PP2A/B55.
-
