Structural basis of p38α regulation by hematopoietic tyrosine phosphatase.

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.

Docking interactions of hematopoietic tyrosine phosphatase with MAP kinases ERK2 and p38α.

Hematopoietic tyrosine phosphatase (HePTP) regulates orthogonal MAP kinase signaling cascades by dephosphorylating both extracellular signal-regulated kinase (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 nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, 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 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.

Resting and active states of the ERK2:HePTP complex.

The MAP kinase ERK2 (ERK2, extracellular signal-regulated kinase 2) 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 show 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.

Sequence-specific backbone ¹H, ¹³C and ¹5N assignments of the 34 kDa catalytic domain of PTPN5 (STEP).

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.

Interaction of kinase-interaction-motif protein tyrosine phosphatases with the mitogen-activated protein kinase ERK2.

The mitogen-activation protein kinase ERK2 is tightly regulated by multiple phosphatases, including those of the kinase interaction motif (KIM) PTP family (STEP, PTPSL and HePTP). Here, we use small angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC) to show that the ERK2:STEP complex is compact and that residues outside the canonical KIM motif of STEP contribute to ERK2 binding. Furthermore, we analyzed the interaction of PTPSL with ERK2 showing that residues outside of the canonical KIM motif also contribute to ERK2 binding. The integration of this work with previous studies provides a quantitative and structural map of how the members of a single family of regulators, the KIM-PTPs, differentially interact with their corresponding MAPKs, ERK2 and p38α.

Strategies to optimize protein expression in E. coli.

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.

Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities.

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.