The lymphoid-specific tyrosine phosphatase (Lyp) has generated enormous interest because a single-nucleotide polymorphism in the gene (PTPN22) encoding Lyp produces a gain-of-function mutant phosphatase that is associated with several autoimmune diseases, including type I diabetes, rheumatoid arthritis, Graves disease, and systemic lupus erythematosus. Thus, Lyp represents a potential target for a broad spectrum of autoimmune disorders. Unfortunately, no Lyp inhibitor has been reported. In addition, little is known about the structure and biochemical mechanism that directly regulates Lyp function. Here, we report the identification of a bidentate salicylic acid-based Lyp inhibitor I-C11 with excellent cellular efficacy. Structural and mutational analyses indicate that the inhibitor binds both the active site and a nearby peripheral site unique to Lyp, thereby furnishing a solid foundation upon which inhibitors with therapeutic potency and selectivity can be developed. Moreover, a comparison of the apo-and inhibitor-bound Lyp structures reveals that the Lypspecific region S 35 TKYKADK 42 , which harbors a PKC phosphorylation site, could adopt either a loop or helical conformation. We show that Lyp is phosphorylated exclusively at Ser-35 by PKC both in vitro and in vivo. We provide evidence that the status of Ser-35 phosphorylation may dictate the conformational state of the insert region and thus Lyp substrate recognition. We demonstrate that Ser-35 phosphorylation impairs Lyp's ability to inactivate the Src family kinases and down-regulate T cell receptor signaling. Our data establish a mechanism by which PKC could attenuate the cellular function of Lyp, thereby augmenting T cell activation.crystal structure ͉ enzyme regulation ͉ Lyp inhibitor ͉ phosphorylation

Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific β-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, β-arrestin-1 ‘reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by 19F-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of β-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific β-arrestin-1 conformations. The identified receptor-phospho-selective mechanism for arrestin conformation and the spacing of the multiple phosphate-binding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors.

The phosphatase of regenerating liver (PRL) phosphatases are implicated in a number of tumorigenesis and metastasis processes. The PRLs are unique among protein-tyrosine phosphatases in that they have extremely low phosphatase activity, a high propensity for trimer formation, and a polybasic region that precedes the C-terminal prenylation motif. To investigate the functional significance of these distinctive biochemical and structural features, we established a cell-based system in which ectopic PRL1 expression increased cell proliferation and migration, whereas knockdown of endogenous PRL1 abrogated these cellular activities. We showed that the intrinsic PRL1 phosphatase activity is obligatory for its biological function. We provided evidence that trimerization may be a general property for all PRL enzymes, and that PRL1 trimer formation is essential for the PRL1-mediated cell growth and migration. This finding indicates a novel mechanism for phosphatase regulation. We further demonstrated that the conserved C-terminal polybasic region is important for specific phosphoinositide recognition by PRL1. Both the polybasic residues and the adjacent prenylation motif are required for proper PRL1 subcellular localization and full biological activity.

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The hyperpolarization-activated cation channels (Ih) play a distinct role in rhythmic activities in a variety of tissues, including neurons and cardiac cells. In the present study, we investigated whether Ca 2؉ can permeate through the hyperpolarization-activated pacemaker channels (HCN) expressed in HEK293 cells and Ih channels in dorsal root ganglion (DRG) neurons. Using combined measurements of whole-cell currents and fura-2 Ca 2؉ imaging, we found that there is a Ca 2؉ influx in proportion to Ih induced by hyperpolarization in HEK293 cells. The Ih channel blockers Cs ؉ and ZD7288 inhibit both HCN current and Ca 2؉ influx. Measurements of the fractional Ca 2؉ current showed that it constitutes 0.60 ؎ 0.02% of the net inward current through HCN4 at ؊120 mV. This fractional current is similar to that of the low Ca 2؉ -permeable AMPA-R (␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) channels in Purkinje neurons. In DRG neurons, activation of Ih for 30 s also resulted in a Ca 2؉ influx and an elevated action potentialinduced secretion, as assayed by the increase in membrane capacitance. These results suggest a functional significance for Ih channels in modulating neuronal secretion by permitting Ca 2؉ influx at negative membrane potentials.