Anita Alexa

Specificity of Linear Motifs that Bind to a Common Mitogen-activated Protein Kinase Docking Groove

Three related kinases use distinct structural features to discriminate between linear docking motifs in potential binding partners. Docking with the Right Partner The mitogen-activated protein kinases (MAPKs) participate in diverse biological processes, such as inflammation and cellular proliferation, and can be divided into the c-Jun N-terminal kinase (JNK), p38, and extracellular signal–regulated kinase (ERK) families. Potential binding partners have short linear “docking” motifs (7 to 17 amino acids) with a loosely defined consensus sequence, and these motifs associate with docking grooves in MAPKs. The docking grooves in the different MAPK families are quite similar in sequence. Garai et al. uncovered the structural features of the docking motifs that enable MAPKs to discriminate between potential binding partners. They used this information to manipulate the specificity of peptides corresponding to docking motifs found in MAPK binding partners—for example, a JNK1-specific peptide was altered so that it also bound to p38α and ERK2. Furthermore, they designed artificial peptides with engineered specificities to a particular MAPK or set of MAPKs. These results provide insight into how MAPKs differentiate between seemingly similar binding partners and could be used to disrupt a specific MAPK binding partner interaction in a targeted fashion. Mitogen-activated protein kinases (MAPKs) have a docking groove that interacts with linear “docking” motifs in binding partners. To determine the structural basis of binding specificity between MAPKs and docking motifs, we quantitatively analyzed the ability of 15 docking motifs from diverse MAPK partners to bind to c-Jun amino-terminal kinase 1 (JNK1), p38α, and extracellular signal–regulated kinase 2 (ERK2). Classical docking motifs mediated highly specific binding only to JNK1, and only those motifs with a sequence pattern distinct from the classical MAPK binding docking motif consensus differentiated between the topographically similar docking grooves of ERK and p38α. Crystal structures of four complexes of MAPKs with docking peptides, representing JNK-specific, ERK-specific, or ERK- and p38-selective binding modes, revealed that the regions located between consensus positions in the docking motifs showed conformational diversity. Although the consensus positions in the docking motifs served as anchor points that bound to common MAPK surface features and mostly contributed to docking in a nondiscriminatory fashion, the conformation of the intervening region between the anchor points mostly determined specificity. We designed peptides with tailored MAPK binding profiles by rationally changing the length and amino acid composition of intervening regions located between anchor points. These results suggest a coherent structural model for MAPK docking specificity that reveals how short linear motifs binding to a common kinase docking groove can mediate diverse interaction patterns and contribute to correct MAPK partner selection in signaling networks.

Mutual protection of microtubule-associated protein 2 (MAP2) and cyclic AMP-dependent protein kinase II against μ-calpain

Phosphorylation by adenosine‐3′,5′‐cyclic monophosphate (cAMP)‐dependent protein kinase (PKA), but not by Ca++‐calmodulin‐dependent protein kinase II (CaMK II), was shown earlier to protect microtubule‐associated protein 2 (MAP2) from cleavage by m‐calpain (Johnson and Foley: J Neurosci Res 34:642–647, 1993). We reinvestigated this phenomenon with the physiologically more relevant μ‐calpain and found a qualitatively similar but quantitatively different picture. We further demonstrate that 1) protection is biphasically dependent on the degree of phosphorylation; 2) Ca++‐phospholipid‐dependent protein kinase (PKC) has about the same effect as PKA; 3) the effects of kinases A and C are not additive; and 4) stripping of native MAP2 from its phosphate content (17.8 ± 2.3 mol/mol) enhances calpainolysis 3.6‐fold.

Novel Cell-Penetrating Calpain Substrate

The calpain enzymes play important roles in numerous processes in the cell. In vivo analysis of calpain activity might be useful for clarification of their role in different diseases. Our early results suggested that a peptide substrate, Dabcyl-TPLKSPPPSPR- EDANS, based on the calpain cleavage sequences is suitable for developing a new cell-penetrating calpain substrate. This conjugate with the Dabcyl and EDANS fluorophores as a FRET pair is specific for calpain even in cell lysate, but unfortunately has poor cell uptake. Therefore, we have modified this sequence by C-terminal elongation with heptaarginine unit possessing cell-penetrating activity. In order to preserve the necessary distance between the two FRET partners, we inserted a Glu residue between the substrate and heptaarginine parts of the peptide. Thus, the cell-penetrating substrate Dabcyl-TPLKSPPPSPRE( EDANS)R 7 was synthesized. This peptide not only retained the substrate property, but was a better substrate of Calpain B enzyme. The cell uptake of the substrate conjugate was studied by fluorescence microscopy and flow cytometry. The results showed that the conjugate enters COS-7 cells more efficiently than the peptide substrate without heptaarginine. The uptake occurs already at low concentration and the compound is distributed homogeneously inside cells. These observations might indicate that this new cell-penetrating substrate could be useful for determining calpain activity in cell lysate or in intact cells of various origins.

Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases

Mitogen‐activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less‐characterized disordered regions. We used a structurally consistent model on kinase‐docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under‐explored part of the human proteome and applied experimental tools specifically tailored to detect low‐affinity protein–protein interactions for their validation in vitro and in cell‐based assays. The combined computational and experimental approach enabled the identification of many novel MAPK‐docking motifs that were elusive for other large‐scale protein–protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase‐mediated partnerships evolved over time. The analysis suggests that most human MAPK‐binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK‐binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.

Structural Basis of Ribosomal S6 Kinase 1 (RSK1) Inhibition by S100B Protein: MODULATION OF THE EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK) SIGNALING CASCADE IN A CALCIUM-DEPENDENT WAY.

Mitogen-activated protein kinases (MAPK) promote MAPK-activated protein kinase activation. In the MAPK pathway responsible for cell growth, ERK2 initiates the first phosphorylation event on RSK1, which is inhibited by Ca2+-binding S100 proteins in malignant melanomas. Here, we present a detailed in vitro biochemical and structural characterization of the S100B-RSK1 interaction. The Ca2+-dependent binding of S100B to the calcium/calmodulin-dependent protein kinase (CaMK)-type domain of RSK1 is reminiscent of the better known binding of calmodulin to CaMKII. Although S100B-RSK1 and the calmodulin-CAMKII system are clearly distinct functionally, they demonstrate how unrelated intracellular Ca2+-binding proteins could influence the activity of the CaMK domain-containing protein kinases. Our crystallographic, small angle x-ray scattering, and NMR analysis revealed that S100B forms a “fuzzy” complex with RSK1 peptide ligands. Based on fast-kinetics experiments, we conclude that the binding involves both conformation selection and induced fit steps. Knowledge of the structural basis of this interaction could facilitate therapeutic targeting of melanomas.

Structural assembly of the signaling competent ERK2–RSK1 heterodimeric protein kinase complex

Significance Signaling pathways often use kinase cascades, but structural characterization of catalytic complexes between heterodimeric kinase pairs has been elusive. For MAPK–MAPKAPK binary complexes, a high-affinity “docking” interaction holds kinase domains proximal within a tethered complex. This heterodimer provided a unique opportunity to shed light on kinase domain–domain contacts that play a role in the assembly of the transient catalytic complex. Starting out from a new precatalytic extracellular signal regulated kinase 2–ribosomal S6 kinase 1 (ERK2–RSK1) crystallographic complex, where the activation loop of the downstream kinase (RSK1) faced the enzymes (ERK2) catalytic site, we used molecular dynamics simulation to show how the catalytic ERK2–RSK1 complex forms. Our findings reveal the dynamic process through which transient, physiologically relevant kinase heterodimers form in a prototypical kinase cascade. Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase–kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzymes (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 “docking” groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they “readjust,” whereas generic kinase domain surface contacts bring them into a catalytically competent state.

Regulation of calpain B from Drosophila melanogaster by phosphorylation

Calpain B is one of the two catalytically competent calpain (calcium‐activated papain) isoenzymes in Drosophila melanogaster. Because structural predictions hinted at the presence of several potential phosphorylation sites in this enzyme, we investigated the in vitro phosphorylation of the recombinant protein by protein kinase A as well as by the extracellular signal‐regulated protein kinases (ERK) 1 and 2. By MS, we identified Ser845 in the Ca2+ binding region of an EF‐hand motif, and Ser240 close to the autocatalytic activation site of calpain B, as being the residues phosphorylated by protein kinase A. In the transducer region of the protease, Thr747 was shown to be the target of the ERK phosphorylation. Based on the results of three different assays, we concluded that the treatment of calpain B with protein kinase A and ERK1 and ERK2 kinases increases the rate of the autoproteolytic activation of the enzyme, together with the rate of the digestion of external peptide or protein substrates. Phosphorylation also elevates the Ca2+ sensitivity of the protease. The kinetic analysis of phosphorylation mimicking Thr747Glu and Ser845Glu calpain B mutants confirmed the above conclusions. Out of the three phosphorylation events tested in vitro, we verified the in vivo phosphorylation of Thr747 in epidermal growth factor‐stimulated Drosophila S2 cells. The data obtained suggest that the activation of the ERK pathway by extracellular signals results in the phosphorylation and activation of calpain B in fruit flies.

Identifying calpain substrates in intact S2 cells of Drosophila.

Calpains are cysteine proteases involved in a number of physiological and pathological processes, yet our knowledge of substrates cleaved in vivo, in intact cells, is scarce. In this work we made an attempt to develop a technique for finding calpain substrates in intact Drosophila Schneider S2 cells. The procedure consists in comparative 2D gelelectrophoresis: three identical samples were treated in different ways: A (control, no addition), B, activated (Ca(2+) and ionomycin added), C, inactivated (additions as in B+specific calpain inhibitor). 2D gel pattern were analyzed by densitometry. Spots showing density relation A>B<<C were identified by mass spectroscopy. In a typical run, 11 candidate substrates were recognized; out of these, four were randomly selected: all four were verified to be calpain substrates, by digestion of the recombinant protein with recombinant calpain.

Theileria highjacks JNK2 into a complex with the macroschizont GPI-anchored surface protein p104

Theileria is a unique apicomplexan parasite capable of transforming its host cell into a disseminating tumour. Constitutive JNK activity characterizes bovine T and B cells infected with T. parva, and B cells and macrophages infected with T. annulata. Here, we show that T. annulata manipulates JNK activation by recruiting JNK2, and not JNK1, to the parasite surface, whereas JNK1 is found predominantly in the host cell nucleus. In silico analysis identified 3 potential JNK-binding motifs in the previously characterized GPI-anchored macroschizont surface protein (p104), and we demonstrate here that JNK2 is recruited to the parasite via physical interaction with p104. A cell penetrating peptide harbouring a p104 JNK-binding motif also conserved in T. parva p104 competitively ablated binding, whereupon liberated JNK2 became ubiquitinated and degraded. Sequestration of JNK2 depended on PKA-mediated phosphorylation of the conserved JNK-binding motif and upon disruption of the p104/JNK2 complex loss of JNK2 resulted in diminished matrigel traversal of T. annulata-transformed macrophages. Loss of JNK2 also resulted in upregulation of small mitochondrial ARF that promoted autophagy consistent with cytosolic sequestration of JNK2 sustaininf not only JNK2, but also nuclear JNK1 levels that combined contribute to both survival and dissemination of Theileria-transformed macrophages. Author Summary Theileria annulata parasites infect and transform their host bovine leukocytes into tumourlike cells that disseminate throughout infected animals causing a widespread disease called tropical theileriosis. Virulence has been ascribed to the parasite’s ability to constitutively activate leukocyte c-Jun N-terminal Kinase (JNK) leading to permanent induction of Matrix Metallo-Proteinase 9 (MMP9) that promotes transformed macrophage dissemination. In attenuated live vaccines JNK-mediated AP-1-driven transcriptional activity is reduced so dampening dissemination. However, in leukocytes JNK exists as two isoforms JNK1 and JNK2 and here, we show for the first time that in T. annulata-transformed macrophages they have different subcellular localisations and perform separate functions. Surprisingly, JNK2 associates with the parasite and is not in the nucleus like JNK1. JNK2 is hijacked by the parasite and sequestered in a complex with a macroschizont surface protein called p104. Upon forced complex dissociation JNK2 gets degraded and its loss negatively affects infected macrophage survival and ability to disseminate.

Dynamic control of RSK complexes by phosphoswitch‐based regulation

Assembly and disassembly of protein–protein complexes needs to be dynamically controlled and phosphoswitches based on linear motifs are crucial in this process. Extracellular signal–regulated kinase 2 (ERK2) recognizes a linear‐binding motif at the C‐terminal tail (CTT) of ribosomal S6 kinase 1 (RSK1), leading to phosphorylation and subsequent activation of RSK1. The CTT also contains a classical PDZ domain‐binding motif which binds RSK substrates (e.g. MAGI‐1). We show that autophosphorylation of the disordered CTT promotes the formation of an intramolecular charge clamp, which efficiently masks critical residues and indirectly hinders ERK binding. Thus, RSK1 CTT operates as an autoregulated phosphoswitch: its phosphorylation at specific sites affects its protein‐binding capacity and its conformational dynamics. These biochemical feedbacks, which form the structural basis for the rapid dissociation of ERK2‐RSK1 and RSK1‐PDZ substrate complexes under sustained epidermal growth factor (EGF) stimulation, were structurally characterized and validated in living cells. Overall, conformational changes induced by phosphorylation in disordered regions of protein kinases, coupled to allosteric events occurring in the kinase domain cores, may provide mechanisms that contribute to the emergence of complex signaling activities. In addition, we show that phosphoswitches based on linear motifs can be functionally classified as ON and OFF protein–protein interaction switches or dimmers, depending on the specific positioning of phosphorylation target sites in relation to functional linear‐binding motifs. Moreover, interaction of phosphorylated residues with positively charged residues in disordered regions is likely to be a common mechanism of phosphoregulation.