MAP kinases regulate essential cellular events, including cell growth, differentiation and inflammation. The solution structure of a complete MAPK–MAPK-regulatory protein complex, p38α–HePTP, was determined, enabling a comprehensive investigation of the molecular basis of specificity and fidelity in MAPK regulation. Structure determination was achieved by combining NMR spectroscopy and small-angle X-ray scattering data with a new ensemble calculation–refinement procedure. We identified 25 residues outside of the HePTP kinase interaction motif necessary for p38α recognition. The complex adopts an extended conformation in solution and rarely samples the conformation necessary for kinase deactivation. Complex formation also does not affect the N-terminal lobe, the activation loop of p38α or the catalytic domain of HePTP. Together, these results show how the downstream tyrosine phosphatase HePTP regulates p38α and provide for fundamentally new insights into MAPK regulation and specificity.

Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T cell antigen receptor (TCR) and are thought to act in a cooperative manner when forming a complex. Here, we studied the spatio-temporal dynamics of the LYP/CSK complex in T cells. We demonstrate that dissociation of this complex is necessary for recruitment of LYP to the plasma membrane, where it down-modulates TCR signaling. Development of a potent and selective chemical probe of LYP confirmed that LYP inhibits T cell activation when removed from CSK. Our findings may explain the reduced TCR-mediated signaling associated with a single nucleotide polymorphism, which confers increased risk for certain autoimmune diseases, including type 1 diabetes and rheumatoid arthritis, and results in expression of a LYP allele that is unable to bind CSK. Our compound also represents a starting point for the development of a LYP-based treatment of autoimmunity.

Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. Herein is described a readily quantifiable novel isopeptidase assay platform consisting of Ub or UBL fused to the reporter enzyme phospholipase A 2 (PLA 2 ). Isopeptidase activity releases PLA 2 , which cleaves its substrate, generating a signal that is linear with deubiquitylase (DUB) concentration and is able to discriminate DUB, deSUMOylase, deNEDDylase, and deISGylase activities. The power and sensitivity of the UBL-PLA 2 assay are demonstrated by its ability to differentiate the contrasting deISGylase and DUB activities of two coronavirus proteases: severe acute respiratory syndrome papain-like protease (SARS-CoV PLpro) and NL63 CoV papain-like protease 2 (PLP2). Furthermore, direct comparisons with the current Ub-7-amino-4-methylcoumarin (Ub-AMC) assay demonstrated that the Ub-PLA 2 assay is an effective tool for characterizing modulators of isopeptidase activity. This observation was expanded by profiling the inhibitory activity of the nonselective isopeptidase inhibitor NSC 632839 against DUBs and deSUMOylases. Taken together, these studies illustrate the utility of the reporter-based approach to measuring isopeptidase activity.Keywords: ubiquitin; deubiquitylase; deSUMOylase; deISGylase; deNEDDylase Supplemental material: see www.proteinscience.orgThe content of most proteins in the cell is governed by the ubiquitin-proteasomal pathway (Hershko and Ciechanover 1998). Ubiquitin (Ub) and ubiquitin-like proteins (UBLs) such as SUMO, NEDD8, and ISG15 regulate proteins by additional mechanisms, for example, intracellular compartmentation, signal transduction, and the regulation of some E3 ligases (Welchman et al. 2005). Degradation of a targeted protein by the ubiquitin system typically involves the concerted action of two to three enzymes (for review, see Hershko and Ciechanover 1998). Typically, polyubiquitylated polypeptides are delivered to the proteasome complex, which hydrolyzes the polypeptide into short oligopeptides and releases free ubiquitin, which is recycled. The process is reversible; ubiquitin, as well as other

Recombinant protein expression in Escherichia coli (E. coli) is simple, fast, inexpensive, and robust, with the expressed protein comprising up to 50 percent of the total cellular protein. However, it also has disadvantages. For example, the rapidity of bacterial protein expression often results in unfolded/misfolded proteins, especially for heterologous proteins that require longer times and/or molecular chaperones to fold correctly. In addition, the highly reductive environment of the bacterial cytosol and the inability of E. coli to perform several eukaryotic post-translational modifications results in the insoluble expression of proteins that require these modifications for folding and activity. Fortunately, multiple, novel reagents and techniques have been developed that allow for the efficient, soluble production of a diverse range of heterologous proteins in E. coli. This overview describes variables at each stage of a protein expression experiment that can influence solubility and offers a summary of strategies used to optimize soluble expression in E. coli.

Summary The MAP kinase p38α is essential for neuronal signaling. To better understand the molecular regulation of p38α we used atomistic and molecular techniques to determine the structural basis of p38α regulation by the two neuronal tyrosine phosphatases, PTPSL/PTPBR7 (PTPRR) and STEP (PTPN5). We show that, despite the fact that PTPSL and STEP belong to the same family of regulatory proteins, they interact with p38α differently and their distinct molecular interactions explain their different catalytic activities. While the interaction of PTPSL with p38α is similar to that of the previously described p38α:HePTP (PTPN7) complex, STEP binds and regulates p38α in an unexpected and new manner. Using NMR and small angle X-ray scattering data we generated a model of the p38α:STEP complex and define molecular differences between its resting- and active-states. Together, these results provide fundamentally new insights into molecular regulation of p38α by key regulatory proteins.

Hematopoietic tyrosine phosphatase (HePTP) regulates orthogonal MAP kinase signaling cascades by dephosphorylating both ERK and p38. HePTP recognizes a docking site (D-recruitment site, DRS) on its targets using a conserved N-terminal sequence motif (D-motif). Using solution NMR spectroscopy and isothermal titration calorimetry (ITC), we compare, for the first time, the docking interactions of HePTP with ERK2 and p38α. Our results demonstrate that ERK2/HePTP interactions primarily involve the D-motif, while a contiguous region called the kinase specificity motif (KIS) also plays a key role in p38α/HePTP interactions. D-motif/DRS interactions for the two kinases, while similar overall, do show some specific differences.

The MAP kinase ERK2 is regulated by numerous phosphatases that tightly control its activity. For example, the hematopoietic tyrosine phosphatase (HePTP) negatively regulates T cell activation in lymphocytes via ERK2 dephosphorylation. However, only very limited structural information is available for these biologically important complexes. Here, we use small angle X-ray scattering combined with EROS ensemble refinement to characterize the structures of the resting and active states of ERK2:HePTP complexes. Our data shows that the resting state ERK2:HePTP complex adopts a highly extended, dynamic conformation that becomes compact and ordered in the active state complex. This work experimentally demonstrates that these complexes undergo significant dynamic structural changes in solution and provides the first structural insight into an active state MAPK complex.

PTPN5 is a protein tyrosine phosphatase that plays an integral role in regulating excitatory postsynaptic activity. The sequence-specific backbone assignments of the murine PTPN5 catalytic domain have been determined based on triple-resonance experiments using uniformly [(2)H,(13)C,(15)N]-labeled protein.