Dissecting the sequence determinants for dephosphorylation by the catalytic subunits of phosphatases PP1 and PP2A

Abstract

The phosphatases PP1 and PP2A are responsible for the majority of dephosphorylation reactions on phosphoserine (pSer) and phosphothreonine (pThr), and are involved in virtually all cellular processes and numerous diseases. The catalytic subunits exist in cells in form of holoenzymes, which impart substrate specificity. The contribution of the catalytic subunits to the recognition of substrates is unclear. By developing a phosphopeptide library approach and a phosphoproteomic assay, we demonstrate that the specificity of PP1 and PP2A holoenzymes towards pThr and of PP1 for basic motifs adjacent to the phosphorylation site are due to intrinsic properties of the catalytic subunits. Thus, we dissect this amino acid specificity of the catalytic subunits from the contribution of regulatory proteins. Furthermore, our approach enables discovering a role for PP1 as regulator of the GRB-associated-binding protein 2 (GAB2)/14-3-3 complex. Beyond this, we expect that this approach is broadly applicable to detect enzyme-substrate recognition preferences.

Targeting an anchored phosphatase-deacetylase unit restores renal ciliary homeostasis

“…As mentioned earlier, we previously identified three secondary PP1 interactive surfaces on AKAP220 ( Schillace and Scott, 1999 ; Schillace et al, 2001 ). Subsequently, the notion of multisite contact between serine/threonine phosphatases and their binding partners has become a standard view ( Hoermann et al, 2020 ). Since it is not technically feasible to remove all four PP1 interactive surfaces on AKAP220 and retain a properly folded protein, we can only conclude that there is a reduced association of the PP1-HDAC6 sub complex in the context of cells expressing AKAP220-ΔPP1.…”

Evolutionary crossroads of cell signaling: PP1 and PP2A substrate sites in intrinsically disordered regions

“…Our laboratory has recently provided evidence that the preferences for basic residues N-terminal of the p-site for PP1 and for pThr over pSer is caused by the catalytic subunit itself [36]. Consequently, not only the regulatory subunits create specificity, but also the surface in close vicinity of the catalytic cleft interacts with AAs Nand C-terminal of the p-site, thereby creating specificity on the motif-level, and also the catalytic cleft creates specificity for pThr over pSer in the cases of PP1c and PP2Ac [31,[36][37][38]. Indeed, also for PP2B recent evidence shows a role of the active site in substrate recognition [39].…”

“…However, this picture for PP1and PP2A substrate specificity is incomplete, since recent advances in phosphatase research combined with the picture from proteomic approaches widen the process for p-site recognition to a contribution from the PP1c and PP2Ac subunit that favors certain motifs surrounding pSer/pThr, similar to the findings for the counteracting kinases: In the holoenzyme scenario the motif context surrounding pSer/pThr played a role for substrate selection [ 29–32 ], and also the preference of the complex of the PP2A catalytic subunit with its B55 regulatory subunit for pThr over pSer overlapped with early observations [ 32–35 ]. Our laboratory has recently provided evidence that the preferences for basic residues N -terminal of the p-site for PP1 and for pThr over pSer is caused by the catalytic subunit itself [ 36 ]. Consequently, not only the regulatory subunits create specificity, but also the surface in close vicinity of the catalytic cleft interacts with AAs N - and C -terminal of the p-site, thereby creating specificity on the motif-level, and also the catalytic cleft creates specificity for pThr over pSer in the cases of PP1c and PP2Ac [ 31 , 36–38 ].…”

“…To take an exemplary look at p-sites in long (≥31 AAs) IDRs [ 45 , 46 ] we analyzed phosphoproteomic datasets from our group [ 36 ] consisting of 1552 pSer/pThr sites. To this end, the full sequences of all corresponding proteins were retrieved from uniprot.org [ 47 ] and analyzed by DISOPRED3 [ 48 ], a reliable algorithm for identification of IDRs [ 49 ], using high-performance computing (HPC, Figure 1A ).…”

“…This approach follows previous definitions for the identification of long IDRs [ 45 , 46 ]. The definition of PP1c or PP2Ac-sensitive sites follows the definition of Supplementary Data 2 in Hoermann et al [ 36 ]. Statistics represent the p-value of a Fisher's exact test carried out in R. ( C and D ) The sequence motif within the categories depicted in Figure 2B were then further analyzed using iceLogo [ 78 ].…”