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CaM


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  • Thrombospondin-1 up-regulates expression of cell adhesion molecules and promotes monocyte binding to endothelium. 15833768

    Expression of cell adhesion molecules (CAM) responsible for leukocyte-endothelium interactions plays a crucial role in inflammation and atherogenesis. Up-regulation of vascular CAM-1 (VCAM-1), intracellular CAM-1 (ICAM-1), and E-selectin expression promotes monocyte recruitment to sites of injury and is considered to be a critical step in atherosclerotic plaque development. Factors that trigger this initial response are not well understood. As platelet activation not only promotes thrombosis but also early stages of atherogenesis, we considered the role of thrombospondin-1 (TSP-1), a matricellular protein released in abundance from activated platelets and accumulated in sites of vascular injury, as a regulator of CAM expression. TSP-1 induced expression of VCAM-1 and ICAM-1 on endothelium of various origins, which in turn, resulted in a significant increase of monocyte attachment. This effect could be mimicked by a peptide derived from the C-terminal domain of TSP-1 and known to interact with CD47 on the cell surface. The essential role of CD47 in the cellular responses to TSP-1 was demonstrated further using inhibitory antibodies and knockdown of CD47 with small interfering RNA. Furthermore, we demonstrated that secretion of endogenous TSP-1 and its interaction with CD47 on the cell surface mediates endothelial response to the major proinflammatory agent, tumor necrosis factor alpha (TNF-alpha). Taken together, this study identifies a novel mechanism regulating CAM expression and subsequent monocyte binding to endothelium, which might influence the development of anti-atherosclerosis therapeutic strategies.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB2150
    Nombre del producto:
    Anti-E-Selectin Antibody, clone P2H3

  • Pull-down of calmodulin-binding proteins. 22297704

    Calcium (Ca(2+)) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca(2+) signaling is mediated through the calcium-binding protein known as calmodulin (CaM). CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca(2+) (Ca(2+)-CaM) as opposed to its Ca(2+)-free state (ApoCaM). While most target proteins bind Ca(2+)-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin, neurogranin (Ng), and certain myosins. These proteins have been shown to play important roles in presynaptic function, postsynaptic function, and muscle contraction, respectively. Their ability to bind and release CaM in the absence or presence of Ca(2+) is pivotal in their function. In contrast, many proteins only bind Ca(2+)-CaM and require this binding for their activation. Examples include myosin light chain kinase, Ca(2+)/CaM-dependent kinases (CaMKs) and phosphatases (e.g. calcineurin), and spectrin kinase, which have a variety of direct and downstream effects. The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca(2+)-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca(2+)-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM. Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca(2+), using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca(2+) on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca(2+)/CaM signaling and proteins that interact with CaM.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-425

  • Phosphorylation-dependent reversible association of Ca2+/calmodulin-dependent protein kinase II with the postsynaptic densities. 9334208

    The association of soluble Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) with postsynaptic densities (PSDs) was determined by activity assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting of the enzyme. Soluble CaM kinase II was autophosphorylated with ATP in the presence of Ca2+ and calmodulin, and then it was incubated with PSDs. Autophosphorylated CaM kinase II was precipitated with PSDs by centrifugation. The kinase that was not autophosphorylated did not precipitate with PSDs. These results indicate that the soluble previously autophosphorylated CaM kinase II associates with PSDs and forms PSD-CaM kinase II complex. A maximum of about 60 microg of soluble CaM kinase II bound to 1 mg of PSD protein under the experimental conditions. Ca2+-independent activity generated by autophosphorylation of the kinase was retained in the PSD-CaM kinase II complex. The CaM kinase II thus associated with PSDs phosphorylated a number of PSD proteins in both the absence and presence of Ca2+. When the CaM kinase II-PSD complex was incubated at 30 degrees C, its Ca2+-independent activity was gradually decreased. This decrease was correlated with dephosphorylation of the kinase and its release from PSD-CaM kinase II complex. These results indicate that CaM kinase II reversibly translocates to PSDs in a phosphorylation-dependent manner.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-881

  • Autophosphorylation of type II CaM kinase in hippocampal neurons: localization of phospho- and dephosphokinase with complementary phosphorylation site-specific antibodies ... 8443414

    We have visualized the distribution of autophosphorylated type II CaM kinase in neural tissue with the use of two complementary antibodies: a monoclonal antibody that binds to the alpha and beta subunits of the kinase only when they are autophosphorylated at threonine-286 (287 in beta) and affinity-purified rabbit antibodies that bind to both subunits only when they are not phosphorylated at these residues. We used these antibodies to double-label organotypic hippocampal cultures, detecting the mouse monoclonal antibody with rhodamine and the rabbit polyclonal antibodies with fluorescein. In double-exposed photographs, the ratios of intensities of the two fluorophores revealed the relative proportion of autophosphorylated and nonphosphorylated kinase in individual neurons throughout the cultures. We found that autophosphorylated and nonphosphorylated kinase are colocalized throughout most neurons rather than segregated within distinct cells or subcellular domains. However, the variations in intensity of the two fluorophores indicated that the proportion of autophosphorylated kinase is consistently higher in neuronal somas than in the neuropil. Incubation of the cultures in Ca2+ free medium dramatically reduced both the level of autophosphorylated kinase detected biochemically and the relative intensity of fluorescent staining with the phosphokinase specific monoclonal antibody. These results support the hypothesis that regulation of Ca(2+)-independent CaM kinase activity in vivo occurs by a dynamic equilibrium between autophosphorylation and dephosphorylation and that this equilibrium is maintained, at varying steady-state levels, in all parts of neurons.
    Tipo de documento:
    Referencia
    Referencia del producto:
    05-533
    Nombre del producto:
    Anti-phospho-CaM Kinase II Antibody, α subunit, (Thr286), clone 22B1

  • Structure, dynamics and interaction with kinase targets: computer simulations of calmodulin. 15023369

    Calmodulin (CaM) is a small protein involved in calcium signaling; among the targets of CaM are a number of kinases, including myosin light chain kinases (MLCK), various CaM-dependent kinases and phosphorylase kinase. We present results of molecular dynamics (MD) simulations of 4-ns length for calmodulin in its three functional forms: calcium-free, calcium-loaded, and in complex with both calcium and a target peptide, a fragment of the smooth muscle MLCK. The simulations included explicit water under realistic conditions of constant temperature and pressure, the presence of counterions and Ewald summation of electrostatic forces. Our simulation results present a more complete description of calmodulin structure, dynamics and interactions in solution than previously available. The results agree with a wide range of experimental data, including X-ray, nuclear magnetic resonance (NMR), fluorescence, cross-linking, mutagenesis and thermodynamics. Additionally, we are able to draw interesting conclusions about microscopic properties related to the protein's biological activity. First, in accord with fluorescence data, we find that calcium-free and calcium-loaded calmodulin exhibit significant structural flexibility. Our simulations indicate that these motions may be described as rigid-body translations and rotations of the N- and C-terminal domains occurring on a nanosecond time scale. Our second conclusion deals with the standard model of calmodulin action, which is that calcium binding leads to solvent exposure of hydrophobic patches in the two globular domains, which thus become ready to interact with the target. Surprisingly, the simulation results are inconsistent with the activation model when the standard definitions of the hydrophobic patches are used, based on hydrophobic clefts found in the X-ray structure of calcium-loaded calmodulin. We find that both experimental and simulation results are consistent with the activation model after a redefinition of the hydrophobic patches as those residues which are actually involved in peptide binding in the experimental structure of the calmodulin-peptide complex. The third conclusion is that the calmodulin-peptide interactions in the complex are very strong and are dominated by hydrophobic effects. Using quasi-harmonic entropy calculations, we find that these strong interactions induce a significant conformational strain in the protein and peptide. This destabilizing entropic contribution leads to a moderate overall binding free energy in the complex. Our results provide interesting insights into calmodulin binding to its kinase targets. The flexibility of the protein may explain the fact that CaM is able to bind many different targets. The large loss of conformational entropy upon CaM:peptide binding cancels the entropy gain due to hydrophobic interactions. This explains why the observed entropic contribution to the binding free energy is small and positive, and not large and negative as expected for a complex with such extensive hydrophobic contacts.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-743

  • An inducible change in Fox-1/A2BP1 splicing modulates the alternative splicing of downstream neuronal target exons. 19762510

    Neuronal depolarization and CaM kinase IV signaling alter the splicing of multiple exons in transcripts for ion channels, neurotransmitter receptors, and other synaptic proteins. These splicing changes are mediated in part by special CaM kinase-responsive RNA elements, within or adjacent to exons that are repressed in the initial phase of chronic depolarization. The splicing of many neuronal transcripts is also regulated by members of the Fox (Feminizing gene on X) protein family, and these Fox targets are also often proteins affecting synaptic activity. We show that Fox-1/Ataxin 2-Binding Protein 1 (A2BP1), a protein implicated in a variety of neurological diseases, can counteract the effects of chronic depolarization on splicing. We find that exon 19 of Fox-1 is itself repressed by depolarization. Fox-1 transcripts missing exon 19 encode a nuclear isoform of Fox-1 that progressively replaces the cytoplasmic Fox-1 isoform as cells are maintained depolarizing media. The resulting increase in nuclear Fox-1 leads to the reactivation of many Fox-1 target exons, including exon 5 of the NMDA receptor 1, that were initially repressed by the high-KCl medium. These results reveal a novel mechanism for the slow modulation of splicing as cells adapt to chronic stimuli: The subcellular localization of a splicing regulator is controlled through its own alternative splicing.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MABE986
    Nombre del producto:
    Anti-PTB Antibody, clone BB7

  • Mapping of calmodulin and Gbetagamma binding domains within the C-terminal region of the metabotropic glutamate receptor 7A. 11395497

    Ca(2+)/calmodulin (Ca(2+)/CaM) and the betagamma subunits of heterotrimeric G-proteins (Gbetagamma) have recently been shown to interact in a mutually exclusive fashion with the intracellular C terminus of the presynaptic metabotropic glutamate receptor 7 (mGluR 7). Here, we further characterized the core CaM and Gbetagamma binding sequences. In contrast to a previous report, we find that the CaM binding motif localized in the N-terminal region of the cytoplasmic tail domain of mGluR 7 is conserved in the related group III mGluRs 4A and 8 and allows these receptors to also bind Ca(2+)/CaM. Mutational analysis of the Ca(2+)/CaM binding motif is consistent with group III receptors containing a conventional CaM binding site formed by an amphipathic alpha-helix. Substitutions adjacent to the core CaM target sequence selectively prevent Gbetagamma binding, suggesting that the CaM-dependent regulation of signal transduction involves determinants that overlap with but are different from those mediating Gbetagamma recruitment. In addition, we present evidence that Gbetagamma uses distinct nonoverlapping interfaces for interaction with the mGluR 7 C-terminal tail and the effector enzyme adenylyl cyclase II, respectively. Although Gbetagamma-mediated signaling is abolished in receptors lacking the core CaM binding sequence, alpha subunit activation, as assayed by agonist-dependent GTPgammaS binding, was not affected. This suggests that Ca(2+)/CaM may alter the mode of group III mGluR signaling from mono- (alpha) to bidirectional (alpha and betagamma) activation of downstream effector cascades.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-765
    Nombre del producto:
    Anti-mGluR4a Antibody

  • CaM Kinase I, active - 042334C

    Tipo de documento:
    Certificado de análisis
    Número de lote:
    042334C
    Referencia del producto:
    14-663
    Nombre del producto:
    CaM Kinase I Protein, active, 10 µg