High‐resolution structural analysis shows how different crystallographic environments can induce alternative modes of binding of a phosphotyrosine peptide to the SH2 domain of Fer tyrosine kinase

Fes and Fes‐related (Fer) protein tyrosine kinases (PTKs) comprise a subfamily of nonreceptor tyrosine kinases characterized by a unique multidomain structure composed of an N‐terminal Fer/CIP4 homology‐Bin/Amphiphysin/Rvs (F‐BAR) domain, a central Src homology 2 (SH2) domain, and a C‐terminal PTK domain. Fer is ubiquitously expressed, and upregulation of Fer has been implicated in various human cancers. The PTK activity of Fes has been shown to be positively regulated by the binding of phosphotyrosine‐containing ligands to the SH2 domain. Here, the X‐ray crystal structure of human Fer SH2 domain bound to a phosphopeptide that has D‐E‐pY‐E‐N‐V‐D sequence is reported at 1.37 å resolution. The asymmetric unit (ASU) contains six Fer‐phosphopeptide complexes, and the structure reveals three distinct binding modes for the same phosphopeptide. At four out of the six binding sites in the ASU, the phosphopeptide binds to Fer SH2 domain in a type I β‐turn conformation, and this could be the optimal binding mode of this phosphopeptide. At the other two binding sites in the ASU, it appears that spatial proximity of neighboring SH2 domains in the crystal induces alternative modes of binding of this phosphopeptide.     

Potential roles for autophosphorylation,kinase activity,and abundance of a CDK-activating kinase (Ee;CDKF;1) during growth in leafy spurge

Leafy spurge (Euphorbia esula L.) is a deep-rooted perennial weed that propagates both by seeds and underground adventitious buds located on the crown and roots. To enhance our understanding of growth and development during seed germination and vegetative propagation, a leafy spurge gene (Accession No. AF230740) encoding a CDK-activating kinase (Ee;CDKF;1) involved in cell-cycle progression was identified, and its function was confirmed based on its ability to rescue a yeast temperature-sensitive CAK mutant (GF2351) and through in vitro kinase assays. Site-directed mutagenesis of Ee;CDKF;1 indicated that two threonine residues (Thr291 and Thr296) were mutually responsible for intra-molecular autophosphorylation and for phosphorylating its substrate protein, cyclin-dependent kinase (CDK). Polyclonal antibodies generated against the Ee;CDKF;1 protein or against a phosphorylated Ee;CDKF;1 peptide [NERYGSL(pT)SC] were used to examine abundance and phosphorylation of CDKF;1 during seed germination and bud growth. The levels of CDKF;1 were lower in dry or imbibed seeds than in germinating seeds or seedlings. Differences in CDKF;1 were also observed during adventitious bud development; small buds appeared to have greater levels of CDKF;1 than large buds. Similar patterns of CDKF;1 expression were detected with either the polyclonal antibody developed using the CDKF;1 protein or the phosphorylated peptide. These results indicated that Thr291 is constitutively phosphorylated in vivo and associated with Ee;CDKF;1 activity. Our results further suggest that a certain level of CDKF;1 activity is maintained in most tissues and may be an important phenomenon for enzymes that regulate early steps in cell-cycle signaling pathways. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Autophosphorylation of cGMP-dependent protein kinase is stimulated only by occupancy of one of the two cGMP binding sites

cGMP-Dependent protein kinase contains, per subunit, 2 binding sites for cGMP. The apparent KD values for site 1 and 2 were 12 and 55 nM. The analogues 8-benzyl-amino-cAMP and N2-monobutyryl-cGMP bind preferentially to site 1 and 2, respectively. Both analogues stimulate autophosphorylation of the enzyme at concentrations at which only half of the phosphotransferase activity of the enzyme is expressed. Complete expression of the phosphotransferase activity requires a high concentration of each analogue and is accompanied by inhibition of the autophosphorylation reactions. It is concluded that occupancy of site 1 or 2 stimulates autophosphorylation while occupancy of both sites prevents autophosphorylation.     

The binding of actin to p38 MAPK and inhibiting its kinase activity in vitro

Ineukaryocyte,themitogen-activatedproteinkinases(MAPKs)playcriticalrolesinmanysignaltransductionprocesses[1].p38signalpathwayisanimportantbranchoftheMAPKs[2].Oneoftheprimaryfunctionsofp38medicatestheinflammatorysignalbyphosphorylatingATF[3].Itisknownthatinhibitingthep38activitycanblockthesignaltransductionofinflammationandsub-sequentlyalleviateinflammatoryresponse[4].Inrecentyears,severalresearchgroupshavetriedtousesomespecificinhibitorsofp38forclinictrial[5—7].IthasbeendemonstratedthatP…     

Identification of residues which regulate activity of the STE20-related kinase hMINK

Activity of the STE20-related kinase hMINK was investigated. hMINK was expressed widely, though not ubiquitously, in human tissues; highest levels being found in haematopoietic tissues but also in brain, placenta, and lung. Mutagenesis revealed that T(191) and Y(193) in the substrate recognition loop of the catalytic domain were critical for kinase activity against exogenous substrates and autophosphorylation. A mutation on T(187) showed reduced enzymatic activity against exogenous substrates but retained autophosphorylation activity. Phosphorylation was confirmed by the use of a phospho-specific T(187) antibody. hMINK activated the JNK signal transduction pathway and optimal JNK activation occurred when the C-terminus was deleted. In addition, overexpression of the C-terminal domain devoid of kinase activity also resulted in significant activation of the JNK pathway. These data suggest that hMINK requires an activation step that dissociates the C terminal, thereby freeing the catalytic domain to interact with substrates. Models for receptor-mediated activation of hMINK are discussed.     

The crystal structure of JNK2 reveals conformational flexibility in the MAP kinase insert and indicates its involvement in the regulation of catalytic activity

c-Jun N-terminal kinase (JNK) 2 is a member of the mitogen-activated protein (MAP) kinase group of signaling proteins. MAP kinases share a common sequence insertion called “MAP kinase insert”, which, for ERK2, has been shown to interact with regulatory proteins and, for p38α, has been proposed to be involved in the regulation of catalytic activity. We have determined the crystal structure of human JNK2 complexed with an indazole inhibitor by applying a high-throughput protein engineering and surface-site mutagenesis approach. A novel conformation of the activation loop is observed, which is not compatible with its phosphorylation by upstream kinases. This activation inhibitory conformation of JNK2 is stabilized by the MAP kinase insert that interacts with the activation loop in an induced-fit manner. We therefore suggest that the MAP kinase insert of JNK2 plays a role in the regulation of JNK2 activation, possibly by interacting with intracellular binding partners.     

Protein kinase C-mediated tyrosine phosphorylation of paxillin and focal adhesion kinase requires cytoskeletal integrity and is uncoupled to mitogen-activated protein kinase activation in human hepatoma cells

Treatment of cultured human hepatoma HepG2 cells with the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), results in an increase in tyrosine phosphorylation of several proteins, including the focal adhesion kinase (FAK) and paxillin using anti-phosphotyrosine Western blotting and immunoprecipitation. However, when cells are in suspension or in the presence of cytochalasin D which disrupts the intracellular network of actin microfilaments, TPA loses its ability to stimulate tyrosine phosphorylation of FAK and paxillin but it still activates mitogen-activated protein kinase (MAPK) and induces PKC translocation from cytosol to the membrane in HepG2 cells. On the other hand, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, blocks TPA-induced MAPK activation but has no effect on TPA-induced tyrosine phosphorylation. Our findings suggest that TPA-induced tyrosine phosphorylation of FAK and paxillin in human hepatoma cells is PKC dependent and requires the integrity of the cell cytoskeleton but is uncoupled to the signal transduction pathway of PKC leading to the translocation of PKC and MAPK activation.     

Novel interactions of ankyrins-G at the costameres: the muscle-specific Obscurin/Titin-Binding-related Domain (OTBD) binds plectin and filamin C

Ankyrins, the adapters of the spectrin skeleton, are involved in local accumulation and stabilization of integral proteins to the appropriate membrane domains. In striated muscle, tissue-dependent alternative splicing generates unique Ank3 gene products (ankyrins-G); they share the Obscurin/Titin-Binding-related Domain (OTBD), a muscle-specific insert of the C-terminal domain which is highly conserved among ankyrin genes, and binds obscurin and titin to Ank1 gene products. We previously proposed that OTBD sequences constitute a novel domain of protein–protein interactions which confers ankyrins with specific cellular functions in muscle. Here we searched for muscle proteins binding to ankyrin-G OTBD by yeast two hybrid assay, and we found plectin and filamin C, two organizing elements of the cytoskeleton with essential roles in myogenesis, muscle cell cytoarchitecture, and muscle disease. The three proteins coimmunoprecipitate from skeletal muscle extracts and colocalize at costameres in adult muscle fibers. During in vitro myogenesis, muscle ankyrins-G are first expressed in postmitotic myocytes undergoing fusion to myotubes. In western blots of subcellular fractions from C2C12 cells, the majority of muscle ankyrins-G appear associated with membrane compartments. Occasional but not extensive co-localization at nascent costameres suggested that ankyrin-G interactions with plectin and filamin C are not involved in costamere assembly; they would rather reinforce stability and/or modulate molecular interactions in sarcolemma microdomains by establishing novel links between muscle-specific ankyrins-G and the two costameric dystrophin-associated glycoprotein and integrin-based protein complexes. These results report the first protein–protein interactions involving the ankyrin-G OTBD domain and support the hypothesis that OTBD sequences confer ankyrins with a gain of function in vertebrates, bringing further consolidation and resilience of the linkage between sarcomeres and sarcolemma.     

Conformational plasticity of the catalytic subunit of protein kinase CK2 and its consequences for regulation and drug design

At the first glance CK2α, the catalytic subunit of protein kinase CK2, is a rigid molecule: in contrast to many eukaryotic protein kinases in CK2α the canonical regulatory key elements like the activation segment occur exclusively in their typical active conformations. This observation fits well to the constitutive activity of the enzyme, meaning, its independence from phosphorylation or other characteristic control factors. Most CK2α structures are based on the enzyme from Zea mays, supplemented by an increasing number of human CK2α structures. In the latter a surprising plasticity of important ATP-binding elements – the interdomain hinge region and the glycine-rich loop – was discovered. In fully active CK2α the hinge region is open and does not anchor the ATP ribose, but alternatively it can adopt a closed conformation, form hydrogen bonds to the ribose moiety and thus retract the γ-phospho group from its functional position. In addition to this partially inactive state human CK2α was recently found in a fully inactive conformation. It is incompatible with ATP-binding due to a combination of a closed hinge and a collapse of the glycine-rich loop into the ATP cavity. These conformational transitions are apparently correlated with the occupation state of a remote docking site located at the interface to the non-catalytic subunit CK2β: if CK2β blocks this site, the fully active conformation of CK2α is stabilized, while the binding of certain small molecule seems to favour the partially and fully inactive states. This observation may be exploited to design effective and selective CK2 inhibitors.     

Autophosphorylation of type 2 casein kinase TS at both its α- and β-subunits: Influence of different effectors

Rat liver casein kinase TS (Ck-TS) having quarternary structure α₂β₂, autophosphorylates at its 25 kDa, β-subunits, incorporating up to 1.2 mol P/mol enzyme. According to their effects on the autophosphorylation pattern the effectors of Ck-TS activity can be grouped into 3 classes: (i) inhibitors, like heparin, which also prevent the autophosphorylation of the β-subunit; (ii) stimulators possessing several amino groups (like spermine) which increase the autophosphorylation at the β-subunit; (iii) stimulators possessing several guanido groups, like protamines and related peptides, which prevent the phosphorylation of the β-subunit, while promoting the autophosphorylation of the 38 kDa α-subunit. In the presence of such polyarginyl effectors the 130 kDa Ck-TS is converted into forms with higher sedimentation coefficient.     

Spinophilin is phosphorylated by Ca2+/calmodulin-dependent protein kinase II resulting in regulation of its binding to F-actin

Spinophilin is a protein phosphatase-1- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We have recently shown that the interaction of spinophilin with the actin cytoskeleton depends upon phosphorylation by protein kinase A. We have now found that spinophilin is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in neurons. Ca(2+)/calmodulin-dependent protein kinase II, located within the post-synaptic density of dendritic spines, is known to play a role in synaptic plasticity and is ideally positioned to regulate spinophilin. Using tryptic phosphopeptide mapping, site-directed mutagenesis and microsequencing analysis, we identified two sites of CaMKII phosphorylation (Ser-100 and Ser-116) within the actin-binding domain of spinophilin. Phosphorylation by CaMKII reduced the affinity of spinophilin for F-actin. In neurons, phosphorylation at Ser-100 by CaMKII was Ca(2+) dependent and was associated with an enrichment of spinophilin in the synaptic plasma membrane fraction. These results indicate that spinophilin is phosphorylated by multiple kinases in vivo and that differential phosphorylation may target spinophilin to specific locations within dendritic spines.     

Modulation of the phosphorylation and activity of calcium/calmodulin-dependent protein kinase II by zinc

Calcium/calmodulin-dependent protein kinase II (CaMPK-II) is a key regulatory enzyme in living cells. Modulation of its activity, therefore, could have a major impact on many cellular processes. We found that Zn(2+) has multiple functional effects on CaMPK-II. Zn(2+) generated a Ca(2+)/CaM-independent activity that correlated with the autophosphorylation of Thr(286), inhibited Ca(2+)/CaM binding that correlated with the autophosphorylation of Thr(306), and inhibited CaMPK-II activity at high concentrations that correlated with the autophosphorylation of Ser(279). The relative level of autophosphorylation of these three sites was dependent on the concentration of zinc used. The autophosphorylation of at least these three sites, together with Zn(2+) binding, generated an increased mobility form of CaMPK-II on sodium dodecyl sulfate gels. Overall, autophosphorylation induced by Zn(2+) converts CaMPK-II into a different form than the binding of Ca(2+)/CaM. In certain nerve terminals, where Zn(2+) has been shown to play a neuromodulatory role and is present in high concentrations, Zn(2+) may turn CaMPK-II into a form that would be unable to respond to calcium signals.     

Coordinate regulation of cadherin and integrin function by the chondroitin sulfate proteoglycan neurocan

N-cadherin and beta1-integrins play decisive roles in morphogenesis and neurite extension and are often present on the same cell. Therefore, the function of these two types of adhesion systems must be coordinated in time and space to achieve the appropriate cell and tissue organization. We now show that interaction of the chondroitin sulfate proteoglycan neurocan with its GalNAcPTase receptor coordinately inhibits both N-cadherin- and beta1-integrin-mediated adhesion and neurite outgrowth. Furthermore, the inhibitory activity is localized to an NH(2)-terminal fragment of neurocan containing an Ig loop and an HA-binding domain. The effect of neurocan on beta1-integrin function is dependent on a signal originating from the cadherin cytoplasmic domain, possibly mediated by the nonreceptor protein tyrosine kinase Fer, indicating that cadherin and integrin engage in direct cross-talk. In the developing chick, neural retina neurocan is present in the inner plexiform layer from day 7 on, and the GalNAcPTase receptor becomes restricted to the inner nuclear layer and the ganglion cell layer (as well as the fiber layer), the two forming a sandwich. These data suggest that the coordinate inhibition of cadherin and integrin function on interaction of neurocan with its receptor may prevent cell and neurite migration across boundaries.     

Use of a semisynthetic epitope to probe histidine kinase activity and regulation

Histidine-aspartic acid phosphotransfer pathways are central components of prokaryotic signal transduction pathways and are also found in many eukaryotes. Tools to study histidine kinases, however, are currently quite limited. In this article, we present a new tool to study histidine-aspartic acid phosphotransfer pathways. We show that many histidine kinases will accept ATPγS as a substrate to form a stable thiophosphohistidine even when they do not form stable phosphohistidines using the natural substrate ATP. An antibody that has previously been used to detect thiophosphorylated serine, threonine, and tyrosine residues is shown to recognize thiophosphohistidine and thiophosphoaspartic acid residues. Histidine kinase autothiophosphorylation is regulated by other protein sensor domains in the same way as autophosphorylation, and thiophosphate is transferred to downstream aspartic acid containing response regulators.     

The leucine 10 residue in the pleckstrin homology domain of ceramide kinase is crucial for its catalytic activity

Ceramide kinase (CERK) converts ceramide (Cer) to ceramide-1-phosphate (C1P), a newly recognized bioactive molecule capable of regulating diverse cellular functions. The N-terminus of the CERK protein encompasses a sequence motif known as a pleckstrin homology (PH) domain. However, little is known regarding the functional roles of this domain in CERK. In this study, we have demonstrated that the PH domain of CERK is essential for its enzyme activity. Using site-directed mutagenesis, we have further determined that Leu10 in the PH domain has an important role in CERK activity. Replacing this residue with a neutral alanine or isoleucine, caused a dramatic decrease in CERK activity to 1% and 29%, respectively, compared to CERK, but had no effect on substrate affinity. The study presented here suggests that the PH domain of CERK is not only indispensable for its activity but also act as a regulator of CERK activity.     

The structure of mitochondrial creatine kinase and its membrane binding properties

The biochemical and biophysical characterization of the mitochondrial creatine kinase (Mi-CK) from chicken cardiac muscle is reviewed with emphasis on the structure of the octameric oligomer by electron microscopy and on its membrane binding properties. Information about shape, molecular symmetry and dimensions of the Mi-CK octamer, as obtained by different sample preparation techniques in combination with image processing methods, are compared. The organization of the four dimeric subunits into the Mi-CK complex as apparent in the end-on projections is discussed and the consistently observed high binding affinity of the four-fold symmetric end-on faces towards many support films and towards each other is outlined. A study on the oligomeric state of the enzyme in solution and in intact mitochondria, using chemical crosslinking reagents, is presented together with the results of a search for a possible linkage of Mi-CK with the adenine nucleotide translocator (ANT). The nature of Mi-CK binding to model membranes, demonstrating that rather the octameric than the dimeric subspecies is involved in lipid interaction and membrane contact formation, is resumed and put into relation to our structural observations. The findings are discussed in light of a possiblein vivo function of the Mi-CK octamer bridging the gap between outer and inner mitochondrial membranes at the contact sites.     

Positive and negative regulation of Raf kinase activity and function by phosphorylation.

Activating and inhibitory phosphorylation mechanisms play an essential role in regulating Raf kinase activity. Here we demonstrate that phosphorylation of C-Raf in the kinase activation loop (residues T491 and S494) is necessary, but not sufficient, for activation. C-Raf has additional activating phosphorylation sites at S338 and Y341. Mutating all four of these residues to acidic residues, S338D/Y341D/T491E/S494D (DDED), in C-Raf results in constitutive activity. However, acidic residue substitutions at the corresponding activation loop sites in B-Raf are sufficient to confer constitutive activity. B-Raf and C-Raf also utilize similar inhibitory phosphorylation mechanisms to regulate kinase activity. B-Raf has multiple inhibitory phosphorylation sites necessary for full kinase inhibition where C-Raf requires only one. We examined the functional significance of these inhibitory and activating phosphorylations in Caenorhabditis elegans lin-45 Raf. Eliminating the inhibitory phosphorylation or mimicking activating phosphorylation sites is sufficient to confer constitutive activity upon lin-45 Raf and induce multi-vulva phenotypes in C.elegans. Our results demonstrate that different members of the Raf family kinases have both common and distinct phosphorylation mechanisms to regulate kinase activity and biological function.