Identification of Substrates of Human Protein-tyrosine Phosphatase PTPN22

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...

supporting

confidence: 77%

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

“…67), and a proteomic approach has been used for the identification of substrates of the PTP, PTPN22, by MS analysis of proteins associated with a substrate-trapping mutant (68). In this study, we demonstrate that a substrate-trapping form of Cdc14 can be used to identify and validate novel substrates, including Bni1, Bnr1, and Kar9.…”

Section: Discussionmentioning

confidence: 93%

Global Analysis of Cdc14 Phosphatase Reveals Diverse Roles in Mitotic Processes

Bloom

Cristea

Procko

et al. 2011

Cdc14 phosphatase regulates multiple events during anaphase and is essential for mitotic exit in budding yeast. Cdc14 is regulated in both a spatial and temporal manner. It is sequestered in the nucleolus for most of the cell cycle by the nucleolar protein Net1 and is released into the nucleus and cytoplasm during anaphase. To identify novel binding partners of Cdc14, we used affinity purification of Cdc14 and mass spectrometric analysis of interacting proteins from strains in which Cdc14 localization or catalytic activity was altered. To alter Cdc14 localization, we used a strain deleted for NET1, which causes full release of Cdc14 from the nucleolus. To alter Cdc14 activity, we generated mutations in the active site of Cdc14 (C283S or D253A), which allow binding of substrates, but not dephosphorylation, by Cdc14. Using this strategy, we identified new interactors of Cdc14, including multiple proteins involved in mitotic events. A subset of these proteins displayed increased affinity for catalytically inactive mutants of Cdc14 compared with the wild-type version, suggesting they are likely substrates of Cdc14. We have also shown that several of the novel Cdc14-interacting proteins, including Kar9 (a protein that orients the mitotic spindle) and Bni1 and Bnr1 (formins that nucleate actin cables and may be important for actomyosin ring contraction) are specifically dephosphorylated by Cdc14 in vitro and in vivo. Our findings suggest the dephosphorylation of the formins may be important for their observed localization change during exit from mitosis and indicate that Cdc14 targets proteins involved in wide-ranging mitotic events.

“…This mutant was used to exclude interactions caused by the substrate trapping activity of LYP (10). We found that ⌬288LYP is able to form a complex with Csk as well as Lck but not with other PTKs in T cells.…”

Section: Binding Of Csk To Lyp Does Not Directlymentioning

confidence: 99%

Autoimmune-associated PTPN22 R620W Variation Reduces Phosphorylation of Lymphoid Phosphatase on an Inhibitory Tyrosine Residue

Fiorillo

Orrù

Stanford

et al. 2010

A missense C1858T single nucleotide polymorphism in the PTPN22 gene recently emerged as a major risk factor for human autoimmunity. PTPN22 encodes the lymphoid tyrosine phosphatase (LYP), which forms a complex with the kinase Csk and is a critical negative regulator of signaling through the T cell receptor. The C1858T single nucleotide polymorphism results in the LYP-R620W variation within the LYP-Csk interaction motif. LYP-W620 exhibits a greatly reduced interaction with Csk and is a gain-of-function inhibitor of signaling. Here we show that LYP constitutively interacts with its substrate Lck in a Csk-dependent manner. T cell receptor-induced phosphorylation of LYP by Lck on an inhibitory tyrosine residue releases tonic inhibition of signaling by LYP. The R620W variation disrupts the interaction between Lck and LYP, leading to reduced phosphorylation of LYP, which ultimately contributes to gainof-function inhibition of T cell signaling.