Biology subjects

Peti, W.

Publications and source records attributed to Peti, W..

8 recordsLinked to original sources

bioRxiv2026

SHP2 binds directly to SOS1 to enable RAS activation

The protein-tyrosine phosphatase SHP2 (PTPN11) regulates growth factor- and cytokine-induced RAS/ERK MAP kinase (MAPK) pathway activation, and aberrant SHP2 function causes developmental disorders and cancer1-5. It is widely believed that the catalytic activity of SHP2 is essential for pathway activation1,4,6-8. This view has shaped our interpretation of how germline PTPN11 mutations cause Noonan Syndrome (NS) and NS with Multiple Lentigines (NS-ML)2,9 and how somatic mutations contribute to myeloproliferative neoplasms and solid tumors1. Here we identify a previously undetected, protein-tyrosine phosphatase (PTP) activity-independent mechanism that revises our understanding of how SHP2 promotes RAS/ERK activation. We find that certain mutations of the nucleophilic cysteine that abolish catalytic activity still promote RAS/ERK pathway activation in normal and neoplastic mammalian cells, zebrafish embryos, and mice. Structural studies show that the SHP2 PTP domain binds directly to the Son of Sevenless 1 (SOS1) Dbl homology (DH) domain. Proximity labeling and super-resolution microscopy demonstrate that SHP2/SOS1 interaction occurs in cells and facilitates SOS1 translocation to the plasma membrane to form clusters. Our results overturn decades of dogma on SHP2 regulation of the RAS/ERK pathway and provide new insights into the mechanism of action of disease-associated PTPN11 mutations.

Cancer Biology↗

bioRxiv2025

Mechanistic and evolutionary insights into a family of aminoacyl-tRNA deacylases that protects against canavanine toxicity

Aminoacyl-tRNA deacylases safeguard the accurate translation of the genetic code by hydrolyzing incorrectly synthesized aminoacyl-tRNAs. Canavanyl-tRNA deacylase (CtdA) was recently shown to protect cells against the toxicity of the non-proteinogenic amino acid canavanine, which is synthesized and accumulated by various plants. In most organisms, canavanine is ligated to tRNAArg, causing translation of arginine codons with canavanine. CtdA prevents canavanine toxicity by hydrolyzing canavanyl-tRNAArg. Here, we investigated the function, structure, substrate specificity, phylogenetic distribution, and evolution of CtdA. We show that CtdA is essential to prevent canavanine cytotoxicity in Salmonella enterica, and its heterologous expression can also rescue Escherichia coli. By determining the structure of CtdA, we identified its putative binding pocket and residues that modulate enzymatic activity and specificity. We also found that CtdA displays a relaxed specificity for the aminoacyl moiety substrate as it hydrolyzes arginyl-tRNAArg. Finally, we showed that despite their structural homology, CtdA and the aminoacyl-tRNA hydrolytic domain of phenylalanyl-tRNA synthetase are functionally and evolutionarily divergent. Collectively, these results substantially expand our understanding of the CtdA family, providing new insights into its structure, function, and evolution. Moreover, this work highlights the diverse mechanisms unique to each organism to ensure faithful translation of the genetic code.

biochemistry↗

bioRxiv2024

Cryo-EM structures of PP2A:B55-Eya3 and PP2A:B55-p107 define PP2A:B55 substrate recruitment

The phosphoprotein phosphatase (PPP) family of ser/thr phosphatases are responsible for the majority of all ser/thr dephosphorylation in cells. However, unlike their kinase counterpart, they do not achieve specificity via phosphosite recognition sequences, but instead bind substrates and regulators using PPP-specific short linear and/or helical motifs (SLiMs, SHelMs). Protein phosphatase 2A (PP2A) is a highly conserved PPP that regulates cell signaling and is a tumor suppressor. Here, we investigate the mechanisms of substrate and regulator recruitment to the PP2A:B55 holoenzyme to define how substrates and regulators engage B55 and understand, in turn, how these interactions direct phosphosite dephosphorylation. Our cryo-EM structures of PP2A:B55 bound to p107 (substrate) and Eya3 (regulator), coupled with biochemical, biophysical and cell biology assays, show that while B55 associates using a common set of interaction pockets, the mechanisms of substrate and regulator binding can differ substantially. This shows that B55-mediated substrate recruitment is distinct from that observed for PP2A:B56 and other PPPs. It also allowed us to identify the core B55 recruitment motif in Eya3 proteins, a sequence we show is conserved amongst the Eya family. Finally, using NMR-based dephosphorylation assays, we also showed how B55 recruitment directs PP2A:B55 fidelity, via the selective dephosphorylation of specific phosphosites. Because of the key regulatory functions of PP2A:B55 in mitosis and DNA damage repair, these data provide a roadmap for pursuing new avenues to therapeutically target this complex by individually blocking a subset of regulators that use different B55 interaction sites.

biochemistry↗

bioRxiv2023

Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation.1 Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases,2 while mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55.3 While the role of kinases in mitotic entry is well-established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited.4 For PP2A:B55, inhibition is achieved by the two intrinsically disordered proteins (IDPs), ARPP19 (phosphorylation-dependent)6,7 and FAM122A5 (inhibition is phosphorylation-independent). Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies both IDPs bind PP2A:B55, but do so in highly distinct manners, unexpectedly leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provides a molecular roadmap for the development of therapeutic interventions for PP2A:B55 related diseases.

biochemistry↗

bioRxiv2021

PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107

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/B55, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55 recruits p107, a pRB-related tumor suppressor and B55 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 "HxRVxxV619-625" short linear motif (SLiM) in p107 as necessary for B55 binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, "p[ST]-P-x(5-10)-[RK]-V-x-x-[VI]-R". Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55 groove, and phosphosite presentation. Altogether these data provide key insights into PP2A/B55 mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55[<] role in multiple cellular processes.

biochemistry↗

bioRxiv2020

The interaction of p38 with its upstream kinase MKK6

Mitogen-activated protein kinase (MAPK; p38, ERK and JNK) cascades are evolutionarily conserved signaling pathways that regulate the cellular response to a variety of extracellular stimuli, such as growth factors and interleukins. The MAPK p38 is activated by its specific upstream MAPK kinases, MKK6 and MKK3. However, a comprehensive molecular understanding of how these cognate upstream kinases bind and activate p38 is still missing. Here, we combine NMR spectroscopy and isothermal titration calorimetry to define the binding interface between full-length MKK6 and p38. We show that p38 engages MKK6 not only via its hydrophobic docking groove, but also helix F, a secondary structural element that plays a key role in organizing the kinase core. We also show that, unlike MAPK phosphatases, the p38 conserved docking (CD) site is much less affected by MKK6 binding. Finally, we demonstrate that these interactions with p38 are conserved independent of the MKK6 activation state. Together, our results reveal differences between specificity markers of p38 regulation by upstream kinases, which do not effectively engage the CD site, and downstream phosphatases, which require the CD site for productive binding.

biophysics↗

bioRxiv2020

1H, 15N and 13C sequence specific backbone assignment of the MAP Kinase Binding Domain of the Dual Specificity Phosphatase 1 and its interaction with the MAPK p38

The sequence-specific backbone assignment of the mitogen-activated protein kinase (MAPK) binding domain of the dual-specificity phosphatase 1 (DUSP1) has been accomplished using a uniformly [13C,15N]-labeled protein. These assignments will facilitate further studies of DUSP1 in the presence of inhibitors/ligands to target MAPK associated diseases and provide further insights into the function of dual-specificity phosphatase 1 in MAPK regulation.

biophysics↗

bioRxiv2017

Choice of PP1 catalytic subunit affects neither the requirement for G-actin nor the insensitivity to Sephin1 of PPP1R15A-regulated eIF2αP dephosphorylation

The integrated stress response (ISR) is regulated by kinases that phosphorylate the subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2P-directed holophosphatase - a therapeutic target in diseases of protein misfolding - is comprised of a regulatory, PPP1R15, and a catalytic, Protein Phosphatase 1 (PP1) subunit. In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in promoting the essential function of eIF2P dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific de-phosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the PPP1R15A-containing PP1 holoenzyme to the [(ochlorobenzylidene)amino]guanidines (Sephin1 or Guanabenz), small molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat-containing region in the PPP1R15A regulatory subunit might influence both the requirement for G-actin by the eIF2P-directed holophosphatase and its sensitivity to inhibitors. Here we report that in the absence of G-actin, PPP1R15A regulatory subunits were unable to accelerate eIF2P dephosphorylation beyond that affected by a catalytic subunit alone, whether PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region and whether paired with native PP1 purified from rabbit muscle, or recombinant PP1 expressed in and purified from bacteria. Furthermore, none of the PPP1R15A-containing PP1c holophosphatases were inhibited by Sephin1 or Guanabenz.

biochemistry↗