Structural Basis of Plasticity in Protein Tyrosine Phosphatase 1B Substrate Recognition ,

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Cdc25 phosphatases are dual specificity phosphatases that dephosphorylate and activate cyclin-dependent kinases (CDKs), thereby effecting the progression from one phase of the cell cycle to the next. Despite its central role in the cell cycle, relatively little is known about the catalytic mechanism of Cdc25. In order to provide insights into the catalytic mechanism of Cdc25, we have performed a detailed mechanistic analysis of the catalytic domain of human Cdc25A. Our kinetic isotope effect results, Bronsted analysis, and pH dependence studies employing a range of aryl phosphates clearly indicate a dissociative transition state for the Cdc25A reaction that does not involve a general acid for the hydrolysis of substrates with low leaving group pK a values (5.45-8.05). Interestingly, our Bronsted analysis and pH dependence studies reveal that Cdc25A employs a different mechanism for the hydrolysis of substrates with high leaving group pK a values (8.68 -9.99) that appears to require the protonation of glutamic acid 431. Mutation of glutamic acid 431 into glutamine leads to a dramatic drop in the hydrolysis rate for the high leaving group pK a substrates and the disappearance of the basic limb of the pH rate profile for the substrate with a leaving group pK a of 8.05, indicating that glutamic acid 431 is essential for the efficient hydrolysis of substrates with high leaving group pK a . We suggest that hydrolysis of the high leaving group pK a substrates proceeds through an unfavored but more catalytically active form of Cdc25A, and we propose several models illustrating this. Since the activity of Cdc25A toward small molecule substrates is several orders of magnitude lower than toward the physiological substrate, cyclin-CDK, we suggest that the cyclin-CDK is able to preferentially induce this more catalytically active form of Cdc25A for efficient phosphothreonine and phosphotyrosine dephosphorylation.

Section: Resultsmentioning

confidence: 76%

The Catalytic Mechanism of Cdc25A Phosphatase

McCain

Catrina

Hengge

et al. 2002

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“…deviation 1.1 Å). Unlike PTP1B, where peptide substrate binding induces the closure of the WPD-loop (encompassing residues 191-199 in Lyp) (29,31), the WPD-loop in both of the Lyp/C227S-peptide complexes exists in the open conformation.…”

Section: 48)mentioning

confidence: 99%

Substrate Specificity of Lymphoid-specific Tyrosine Phosphatase (Lyp) and Identification of Src Kinase-associated Protein of 55 kDa Homolog (SKAP-HOM) as a Lyp Substrate

Yu¹,

Chen²,

Zhang

et al. 2011

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A missense single-nucleotide polymorphism in the gene encoding the lymphoid-specific tyrosine phosphatase (Lyp) has been identified as a causal factor in a wide spectrum of autoimmune diseases. Interestingly, the autoimmune-predisposing variant of Lyp appears to represent a gain-of-function mutation, implicating Lyp as an attractive target for the development of effective strategies for the treatment of many autoimmune disorders. Unfortunately, the precise biological functions of Lyp in signaling cascades and cellular physiology are poorly understood. Identification and characterization of Lyp substrates will help define the chain of molecular events coupling Lyp dysfunction to diseases. In the current study, we identified consensus sequence motifs for Lyp substrate recognition using an "inverse alanine scanning" combinatorial library approach. The intrinsic sequence specificity data led to the discovery and characterization of SKAP-HOM, a cytosolic adaptor protein required for proper activation of the immune system, as a bona fide Lyp substrate. To determine the molecular basis for Lyp substrate recognition, we solved crystal structures of Lyp in complex with the consensus peptide as well as the phosphopeptide derived from SKAP-HOM. Together with the biochemical data, the structures define the molecular determinants for Lyp substrate specificity and provide a solid foundation upon which novel therapeutics targeting Lyp can be developed for multiple autoimmune diseases.Protein-tyrosine phosphorylation-mediated signal transduction is essential for a wide range of eukaryotic functions, including cell proliferation, differentiation, migration, apoptosis, and the immune responses (1). This signaling mechanism is often dysregulated in human diseases, due to aberrant activity of the enzymes involved in the regulation of protein tyrosine phosphorylation status (1, 2). Thus, a comprehensive understanding of the physiological roles of protein tyrosine phosphorylation and how this process is abrogated in the pathogenesis of human diseases must necessarily include characterization of proteintyrosine phosphatases (PTPs), 4 in addition to protein-tyrosine kinases.The lymphoid-specific tyrosine phosphatase (Lyp) has garnered tremendous interest due to the observation that a missense C1858T single nucleotide polymorphism in the gene (PTPN22) encoding Lyp is associated with multiple autoimmune disorders, including type I diabetes (3), rheumatoid arthritis (4, 5), Graves disease (6), and systemic lupus erythematosus (7). Lyp expression is restricted to human T cells, B cells, and macrophages (8). Lyp exerts an inhibitory effect on the proximal T cell receptor signaling pathways, probably through dephosphorylation of the Lck and ZAP-70 kinases (9 -11). The C1858T single nucleotide polymorphism changes Arg 620 into a Trp within the first Pro-rich region in the C terminus of Lyp, leading to loss of Lyp binding to the Src homology 3 domain of the Src C-terminal kinase (Csk) (3, 4). Importantly, the autoimmune-predisposing variant of...

“…2D). Although PTP1B has been shown to dephosphorylate a wide variety of synthetic peptides in vitro (18), it displayed no measurable activity against the H2A.X phospho-Tyr-142 peptide. Overall, these data suggest that EYA2 and EYA3 have the potential to exhibit selectivity in the dephosphorylation of phosphoTyr-142 of histone H2AX.…”

mentioning

confidence: 99%

Dephosphorylation of the C-terminal Tyrosyl Residue of the DNA Damage-related Histone H2A.X Is Mediated by the Protein Phosphatase Eyes Absent

Krishnan

Jeong

Jung

et al. 2009

131

146

In mammalian cells, the DNA damage-related histone H2A variant H2A.X is characterized by a C-terminal tyrosyl residue, Tyr-142, which is phosphorylated by an atypical kinase, WSTF. The phosphorylation status of Tyr-142 in H2A.X has been shown to be an important regulator of the DNA damage response by controlling the formation of ␥H2A.X foci, which are platforms for recruiting molecules involved in DNA damage repair and signaling. In this work, we present evidence to support the identification of the Eyes Absent (EYA) phosphatases, protein-tyrosine phosphatases of the haloacid dehalogenase superfamily, as being responsible for dephosphorylating the C-terminal tyrosyl residue of histone H2A.X. We demonstrate that EYA2 and EYA3 displayed specificity for Tyr-142 of H2A.X in assays in vitro. Suppression of eya3 by RNA interference resulted in elevated basal phosphorylation and inhibited DNA damage-induced dephosphorylation of Tyr-142 of H2A.X in vivo. This study provides the first indication of a physiological substrate for the EYA phosphatases and suggests a novel role for these enzymes in regulation of the DNA damage response.Unlike kinases, which are derived from a common ancestor, the opposing phosphatases have evolved in separate, structurally distinct families. In fact, a variety of catalytic mechanisms have been harnessed to facilitate protein dephosphorylation. The protein-Ser/Thr phosphatases, such as the PPP and PPM families, mediate dephosphorylation by using two metal ions at the active site, activating a water molecule for nucleophilic attack on the substrate phosphate in a single-step reaction (1). The family of Cys-dependent protein-tyrosine phosphatases (PTPs) 4 utilizes a two-step catalytic mechanism involving an essential nucleophilic cysteinyl residue, which forms a Cysphosphate intermediate (2). More recently, a third family of Asp-dependent phosphatases belonging to the haloacid dehalogenase (HAD) superfamily (3) has been gaining prominence.Various HAD phosphatases have been linked to fundamental aspects of control of cell function. One of the best characterized is FCP1, which is an important regulator of transcription through dephosphorylation of the C-terminal domain of RNA polymerase II (4). Chronophin has been shown to dephosphorylate Ser-3 in cofilin and thereby function as a regulator of the actin cytoskeleton (5). More recently, a HAD phosphatase known as Dullard has been shown to dephosphorylate the phosphatidic acid phosphatase lipin, functioning in a phosphatase cascade that regulates nuclear membrane biogenesis (6). In these cases, the HADs are functioning as protein-Ser/Thr phosphatases. However, there is now also an example of a member of the HAD superfamily with the capacity to dephosphorylate phosphotyrosyl residues in substrates.Eyes Absent (EYA) was identified initially as a component of a network of transcription regulators, the retinal determination gene network that is responsible for eye development in Drosophila. It is now known to be involved in tissue and organ developme...